Journal

of
Dentistry
Journal of Dentistry 27 (1999) 557–564
www.elsevier.com/locate/jdent

Human pulp response after an adhesive system application in deep cavities

J. Hebling a,*, E.M.A. Giro b, C.A.S. Costa a,1
a
University of Michigan School of Dentistry, 821 Mt. Pleasant Avenue, Ann Arbor, MI 48103, USA
b
Araraquara Dental School, State of São Paulo University, Rua Humaitá, 1680-CEP: 14801-903-CP: 331, Araraquara, S.P. Brazil
Received 5 August 1998; received in revised form 22 February 1999; accepted 26 February 1999

Abstract
Objectives: To evaluate the pulpo-dentin complex response to a dentin adhesive application in deep cavities performed in human teeth.
Methods: Deep class V cavities were prepared on the buccal surface of 46 premolars. The remaining dentin of the axial wall received 10%
phosphoric acid and dentin adhesive (group DA), or was protected before the acid and dentin adhesive application with calcium hydroxide
cement (group CH). Half of the teeth, which received the acid application directly over the axial wall, were contaminated prior to the
procedures with dental plaque collected from the patient’s own teeth (group DAC). The plaque was placed on the dentin for 5 min and then
the cavity was washed. All teeth were restored with a light-cured composite resin. The teeth were extracted after 7, 30 or 60 days and
prepared according to normal histologic techniques. Serial sections were stained with H/E, Masson’s trichrome and Brown & Brenn
technique for demonstration of bacteria.
Results: The histopathologic evaluation showed that in groups DA and DAC, the inflammatory response was more evident than in group
CH. Also, the intensity of the pulp reaction increased as the remaining dentin thickness decreased. There was no statistical difference in the
inflammatory response between the groups DA and DAC.
Conclusion: Based on the experimental conditions, we concluded that the All Bond 2 adhesive system, when applied on dentin in deep
cavities, showed an acceptable biocompatibility. However, the intensity of the pulpo-dentin complex response depends on the remaining
dentin thickness. q 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Biocompatibility; Adhesive resin; Dentin; Human tooth

1. Introduction resins can be associated with intense irritation of pulp [3,4].

Some studies have shown enlargement and congestion of
For many years the chemical action of dental materials blood vessels, odontoblast displacement or dentin matrix
was believed to be the main factor responsible for necrosis deposition, following the application of these materials
and pulp irritation. However, several studies [1,2] have [3,4].
reported the association between the presence of bacteria Many dentin-bonding systems require application of acid
on the cavity walls beneath the filling material and pulp conditioners to remove the smear layer from cavity walls and
inflammation. These authors suggest that the pulp response to promote the superficial demineralization of dentin substrate.
does not depend on the type of material applied over the These products can alter the physical and chemical dentin
pulpo-dentin complex, but upon the capability of filling properties, facilitating its penetration through the dentin
materials to prevent bacterial microleakage [1,2]. tubules. Although the pulp hydrostatic pressure can limit the
Although resins may have adequate physical properties, diffusion through dentin, it cannot avoid its occurrence [5].
in vivo studies have shown that the clinical application of Since studies have demonstrated a possible chemical
toxicity of adhesive systems components [6,7], the purpose
of the present work was to evaluate in human teeth the
* Corresponding author. Department of Orthodontics and Pediatric
Dentistry, University of São Paulo State-UNESP, Rua Humaitá, 1680-
pulpo-dentin complex response to a dentin adhesive system
CEP: 14801-903, CP: 331, Araraquara, São Paulo, Brazil. Tel.: 1 232- application in deep cavities.
1233; fax: 1 222-4823.
E-mail addresses: infantil@black.foar.unesp.br (J. Hebling), hebcosta
@umich.edu (C.A.S. Costa)
1
Dr. C.A.S. Costa, University of Michigan/School of Dentistry, Depart- 2. Materials and methods
ment of Oral Pathology, 1011 N. University, 5th floor, room 5224, Ann
Arbor, MI 48109-1078, USA, Tel./fax: 734-995-3216. Forty-six human premolar teeth, free of fillings, cervical
0300-5712/99/$ - see front matter q 1999 Elsevier Science Ltd. All rights reserved.
PII: S0300-571 2(99)00034-2
558 J. Hebling et al. / Journal of Dentistry 27 (1999) 557–564

Table 1 Table 3
Teeth distribution according to groups and experimental periods Tissue disorganization

Group Description Periods Total Tissue Characterization

7 days 30 days 60 days disorganization

DA Cavity 6 6 6 18 Score 0 Normal tissue

preparation 1 Score 1 Odontoblastic layer disorganized but central pulp
dentin normal
adhesive 1 resin Score 2 Total disorganization of the pulp tissue morphology
DAC Cavity 6 6 4 16 Score 3 Pulp necrosis
preparation 1
contamination 1
dentin
adhesive 1 resin the burs were replaced to avoid excessive heating. The
CH Cavity 4 4 4 12 teeth were divided into three groups (Table 1) and the
preparation 1 same dentin adhesive system, All Bond 2 (Bisco, Itasca,
calcium IL, USA), was used for all teeth. In the experimental groups
hydroxide
(groups DA and DAC), enamel and dentin were directly
cement 1 dentin
adhesive 1 resin etched for 15 s using 10% phosphoric acid followed by
application of five consecutive layers of primer which
were dried slightly, and finally by the adhesive, which
abrasion and caries and scheduled to be removed for ortho- was light cured for 20 s. For group DAC, before the acid
dontic purposes, were selected from patients ranging in age etching the axial wall was covered for 5 min with plaque
from 12 to 15 years old. The patients signed consent forms collected from the same patient during the same clinical
authorizing utilization of the teeth after the patients had procedure. Then, the plaque was removed with washing
received necessary explanations. Before the start of the and the succeeding steps were the same as in groups DA
operative protocol, every tooth was radiographically exam- and CH. In the control group (group CH), before the acid
ined for periapical conditions using X-ray equipment etch the axial wall was protected with hard calcium hydro-
(Gnatus Mod. XR 6010, Ribeirão Preto, S.P., Brazil) xide cement (Hidro C—Dentsply, Mildford, DE, USA) and
adjusted to 15 mHs, 60 KV, 60 Hz and 0.6 s of exposure. the following procedures were exactly the same. All cavities
After local anesthesia, using an anesthetic with vasocon- were filled with Z100 hybrid resin (3M, St. Paul, MN, USA)
strictor, and rubber dam isolation, each tooth was polished in two incremental layers, with 40 s of light curing (Curing
with a rubber cup and prophylactic paste at low speed and light XL 1000—3M, St. Paul, MN, USA) to each layer. The
cleaned with 70% alcohol. With a number 1091 diamond light had its intensity checked immediately before each clin-
bur (KG Sorensen, Barueri, S.P., Brazil), which had its ical section, using a radiometer (Demetron Research
active tip previously limited to 2.5 mm with a resin stop Corporation—model 100P/N 10503, Danbury, CT, USA),
to allow minimum differences among cavities depth, class and the intensity was no less than 400 mW/cm 2. When
V cavities were prepared at high speed with copious water necessary, the cervical excesses were removed using a
irrigation in the cervical region on the buccal surface. The diamond bur with small grid at high speed.
final cavity dimensions were 3 mm length, 2.5 mm depth After 7, 30 or 60 days, the teeth were extracted under
and 1.5 mm width. The axial wall was smoothed using a local anesthesia. Immediately their roots were sectioned
bur at low speed, and finally the cavity was vigorously halfway between the CEJ and the root tip with a high
washed and cleaned. After each four cavity preparations, speed handpiece under intensive water spray to allow pene-
tration of the fixative solution. The teeth were stored for
Table 2 48 h in 10% Karnovsky’s fixative in containers labeled
Inflammatory cell response with random numbers. Then, the specimens were deminer-
alized with 10% EDTA in Karnovsky’s fixative at pH 7.2 at
Inflammatory Characterization
108C, prepared according to normal histologic techniques
cell response
and embedded in paraffin.
Score 0 None or a few scattered inflammatory cells present in Five micrometer thick sections were cut (“820” Spencer
the pulp area corresponding to the axial wall, Microtome, Carson, CA, USA) parallel to the main vertical
characteristic of normal tissue axis of the tooth through the cavity preparation. The slides
Score 1 Slight inflammatory cell infiltrate with
polymorphonuclear (PMNs) or mononuclear
were stained with hematoxylin and eosin (H/E), Masson’s
leukocytes (MNLs) trichrome and Brown & Brenn (B&B) technique. All
Score 2 Moderate inflammatory cell infiltrate involving the sections were evaluated blindly for five histologic features:
coronal pulp inflammatory cellular response, tissue disorganization,
Score 3 Severe inflammatory cell infiltrate involving the reactionary dentin formation, bacteria and the remaining
coronal pulp or characterizing abscess
dentin thickness (RDT). The intensity of the pulp response
 J. Hebling et al. / Journal of Dentistry 27 (1999) 557–564 559

Table 4 in which the RDT had 291 and 280 mm, respectively, there
Reactionary dentin formation could be seen a persistent moderate inflammatory response,
Reactionary Characterization consisting predominantly of macrophages and hydropic
dentin formation degeneration of pulp cells (Fig. 4(B)).
At the last period of evaluation, only one specimen of
Score 0 Absence
group DA presented persistent pulp tissue disorganization
Score 1 Modest hard tissue deposition beneath the axial wall
Score 2 Moderate hard tissue deposition beneath the axial wall associated with moderate inflammatory response mediated
Score 3 Intense hard tissue deposition beneath the axial wall by macrophages. All other specimens showed pulp repair
with a thin layer of reactionary dentin laid down on pulp
tissue subjacent to cavity floor (Fig. 5(A)). However, in
was evaluated by light microscope (Carl Zeiss 62774, Ober-
specimens from group CH, the pulp tissue exhibited normal
kachen-West Germany), using the criteria defined in the
histologic characteristics and no reactionary dentin (Fig.
Tables 2–5. The RDT between the cavity floor and pulp
5(B)).
tissue (Fig. 1(A)) was measured for each specimen using a The application of ANOVA analysis (Table 8) to the data
light microscope (DIASTAR—Cambridge Instruments,
present in Table 7 did not show those both factors, Group or
Buffalo, NY, USA) adapted to a video camera (DXC—
Period, and the interaction between them were significant
107A/107AP—Sony electronics Inc., Tokyo, Japan), and
(p , 0.05). Thus, the RDT mean among the groups for each
to a microcomputer and software (Mocha, Jondel Scientific,
period could be considered the same. The results showed an
San Rafael, CA, USA).
inverse relationship between the RDT and the pulp inflam-
For statistical analysis, ANOVA and Fisher’s test were
matory response. Therefore, the intensity of the pulp
performed to determine if there was a statistically signifi-
response increased as the RDT decreased.
cant difference (p , 0.05) among ranked groups and peri- The statistical model application of the criteria evaluated
ods.
showed a significant difference only among groups when the
inflammatory cell response was analyzed (p , 0.05). The
Fisher’s analysis showed that the groups DA and DAC (Fig.
3. Results 6(A)) presented greater inflammatory responses than group
CH but there was no significant difference between the DA
The scores observed for every criterion according to and the DAC groups. The Fig. 6(A) showed that the inflam-
groups and periods are shown on Table 6 and the matory response continued from 7 to 60 days time periods.
individual 1 mean RDT values are presented in Table 7. For both tissue disorganization (Fig. 6(B)) and bacterial
Overall, the histological features showed that the pulp presence (Fig. 6(D)), only the treatments presented signifi-
response for groups DA and DAC were quite similar. In cant differences (p , 0.05). The Fisher’s analysis showed
one specimen for group DA at 7 day period, the RDT was significant differences between groups DA and DAC when
240 mm. In this specimen, odontoblasts had been displaced compared with group CH for tissue disorganization. For
into the dentinal tubules and inflammatory response with bacterial presence, group DAC presented scores significantly
neutrophils and macrophages occurred in the pulp tissue
adjacent to cavity floor (Fig. 1(B)). The hyaline alteration
of extracellular matrix associated with hydropic degenera-
tion occurred in the pulp horn (Figs. 1(B), 2(A) and (B),
3(A)). In this specimen, long resin tags formed within the
dentinal tubules, where globules of resin were seen floating
through the dentinal tubules (Fig. 3(B)).
At 30 days, a thin layer of reactionary dentin was present
in the pulp tissue area subjacent to cavity floor (Fig. 4A).
Although in one specimen of each of groups DA and DAC,

Table 5
Stained bacteria

Bacteria Characterization
presence

Score 0 Absence
Score 1 Presence of stained bacteria along the cavity lateral walls Fig. 1. (A) Relationship between cavity preparation and pulp tissue (X).
Score 2 Presence of stained bacteria along the cavity lateral and H&E; 56 ×. (B) All Bond 2–7 days. Remaining dentin between cavity floor
axial walls and pulp tissue was 240 mm thick (X). The odontoblast layer is disrupted
Score 3 Presence of stained bacteria along the cavity walls and and the cells were displaced into the dentinal tubules (vertical arrow).
within the cut dentin tubules Moderate number of inflammatory cells is present in the area previously
occupied by odontoblasts (horizontal arrow). H&E; 125 ×.
560 J. Hebling et al. / Journal of Dentistry 27 (1999) 557–564

Table 6
Number of teeth for each score according to groups and periods

Histopatologic event Periods 7 days 30 days 60 days Total

Scores 0 1 2 3 0 1 2 3 0 1 2 3

Groups
Inflammatory cell response DA 4 1 1 0 3 2 1 0 1 4 1 0 18
DAC 0 6 0 0 0 5 1 0 1 3 0 0 16
CH 2 2 0 0 4 0 0 0 4 0 0 0 12

Tissue Disorganization DA 2 3 1 0 0 5 1 0 1 4 1 0 18
DAC 2 4 0 0 1 4 1 0 0 4 0 0 16
CH 2 2 0 0 4 0 0 0 4 0 0 0 12

Bacterial presence DA 4 1 0 0 2 2 1 1 3 1 2 0 18
DAC 1 3 1 1 2 3 1 0 0 1 3 0 16
CH 3 1 0 0 1 2 1 0 1 3 0 0 12

Dentin formation DA 6 0 0 0 4 2 0 0 2 4 0 0 18
DAC 6 0 0 0 2 3 1 0 2 2 0 0 16
CH 4 0 0 0 4 0 0 0 4 0 0 0 12

higher than groups DA and CH. Fig. 6(B) and (C) shows the cavity configuration. Although many authors believe that
interaction between groups and periods for these two pulp tissue response is caused only by the presence of
criteria, respectively. In contrast, in the Fisher’s analysis, bacteria, several in vitro studies have demonstrated that
both Group and Period exerted influence over reactionary resin monomers diffuse through dentin tubules [8] and
dentin formation (p , 0.05), where groups DA and DAC cause cytotoxicity [9].
presented more reactionary dentin formation (Fig. 6(C)) Some studies have demonstrated an inverse relationship
than group CH, with increase in this formation with time. between the remaining dentin thickness and the inflamma-
tory pulp response [3,4], which agree with the results
observed in the present work. Our findings agree with the
results observed by Hanks et al. [10], who demonstrated that
4. Discussion
0.5 mm of dentin seems enough to provide protection to
The divergence of results observed in the literature pulp tissue against toxic substances diffusion. Pashley [11]
concerning the biocompatibility of adhesive systems can
be explained by facts such as adhesive chemistry, methods Table 7
Remaining dentin thickness (mm)
of application, operator consistency, dentin wetting and
Group Specimens Periods
7 days 30 days 60 days

DA 1 240 227 875

2 732 291 1013
3 936 582 223
4 528 165 355
5 462 458 780
6 479 542 442
Mean 562.8 377.5 614.7

DAC 1 299 517 470

2 292 629 267
3 576 432 691
4 875 609 564
5 661 280 —
6 479 288 —
Mean 530.3 459.2 498.0

Fig. 2. (A) All Bond 2–7 days. Inflammatory pulp reaction underlying the CH 1 853 770 737
dentinal tubules related with cavity floor. Note the cells displaced into the 2 857 615 513
dentinal tubules (arrow) and the hyaline alteration of extracellular matrix 3 736 790 353
adjacent to inflammatory pulp reaction (X). H&E; 160 ×. (B) high magni- 4 528 372 369
fication of Fig. 2(A) showing the central zone of pulp tissue which demon- Mean 743.5 636.7 493.0
strate the hyaline alteration of extracellular matrix (X). H&E; 210 ×.
 J. Hebling et al. / Journal of Dentistry 27 (1999) 557–564 561

Fig. 3. (A) All Bond 2–7 days. Higher magnification of Fig. 2(A), demon- Fig. 5. (A) All Bond 2–60 days. Reactionary dentin formation with odon-
strating the aspiration of cells into the dentinal tubules associated with toblast layer underlying. Note a discrete disorganization of superficial pulp
inflammatory response of underlying pulp tissue. Note a large number of tissue but with no remarkable inflammatory response. H&E; 148 ×. (B)
macrophages and neutrophils in degeneration. H&E; 240 ×. (B) All Bond calcium hydroxide–7 days. Pulp tissue showing the odontoblast layer
2–7 days. Presence of hybrid layer on cavity floor (X) and long resin tags with no deposition of reactionary dentin. A slight disorganization of super-
into the dentinal tubules (vertical arrow). Note fragments of resin diffusing ficial pulp zones can be seen in spite of absence of inflammatory pulp
through the dentinal tubules (head arrow). H&E; 210 ×. reaction. H&E; 148 ×.

higher dentinal permeability. This mechanism is enhanced

reported that in exposed dentin, the outward fluid flow
by the smear layer removal and structural alteration of the
through tubules is a first line of defense against the inward
dentin tissue caused by the acid application.
diffusion of noxious substances. Dentinal fluid also contains
plasma proteins (albumin and globulin) which may bind or Excessive heating of the cavity preparation can also cause
a pulp inflammatory response as well as odontoblast
agglutinate some materials, and thus serve in a protective
displacement. However, the presence of pulp cells within
role [11]. This inverse relationship can be explained by
the dentinal tubules was observed only at 7 days on groups
studies, which have shown an increased dentin permeability
DA and DAC in specimens showing a small RDT between
correlating with lower RDT [10,11]. Reduced dentin thick-
cavity floor and pulp tissue. As this finding was not seen in
ness results in an increased diameter and a number of the
specimens belonging to group CH, the possibility of
dentinal tubules close the pulp tissue and consequently
heating during the cavity preparation does not explain the
phenomenon. In accordance with Stanley [12], pulpal
inflammation may be due to the acid solution applied
directly on the dentin tissue. Most dentin acid conditioners
are hypertonic and their application on dentin remove the
smear layer and decalcify the peritubular dentin, causing
increased dentinal tubule diameter [11]. These procedures
increase the dentin permeability drastically and seem to
cause cell displacement. In this way, acid conditioners can
contribute to the immediate pulp response. Also, the hydro-
philic component of adhesive resins applied on dentinal
tissue may promote outward fluid shifts. The fast change
of fluid shifts might contribute to odontoblast displacement
into dentin tubules [11], as seen in the present study.

Table 8
ANOVA analysis
Fig. 4. (A) All Bond 2–30 days. Detail of pulp tissue related to cavity
preparation where the remaining dentin thickness between cavity floor and DF Sum of squares Mean square f-value p-value
pulp tissue is 584 mm. Observe the reactionary dentin layer deposited (B),
slight disorganization of superficial pulp tissue (X) and large number of Group 2 123985.36 61992.68 1.360 a 0.269
small blood vessels. H&E; 160 ×. (B) All Bond 2–30 days. Pulp tissue Period 2 115644.37 57822.18 1.268 a 0.293
adjacent to cavity preparation with 291 mm of remaining dentin thickness. Gr. × Pr. 4 207020.42 51755.10 1.135 a 0.355
Note the reactionary dentin formation (B) with hydropic degeneration of Residual 37 140687.70 45597.07
adjacent pulp cells (arrow). Persistent chronic inflammation can be seen
beside the pulp cell and blood vessel proliferation. H&E; 160 ×.
a
Not significant values, significance level p , 0.05.
562 J. Hebling et al. / Journal of Dentistry 27 (1999) 557–564

Fig. 6. Fisher’s analysis showing interaction between groups and periods for the criteria evaluated: (A) inflammatory cell response; (B) tissue disorganization;
(C) reactionary dentin; and (D) bacteria.

Reactionary dentin is laid down by primary odontoblast inflammatory response when the authors applied All Bond 2
cells in response to specific stimuli, which is not strong on to deep cavities prepared in human teeth. The authors
enough to kill those primary pulp cells [13]. Consequently, demonstrated by TEM the movement of resin particulates
the reactionary dentin deposition seems to be the result of through dentinal tubules. The presence of resin components
slight to moderate irritation and can be inhibited when in the pulp tissue promoted a persistent chronic inflamma-
severe irritation (toxicity) is present. Thus, there is a gradi- tory response, with reaction of macrophages or giant cells.
ent effect related to concentration of the irritant. Therefore, The irritating action of resin particulates, which are not
while the presence of reactionary dentin may be the soluble and cannot be digested, may continue for a long
evidence of the presence of an irritating agent [3], it may time, as seen in this present study, and is proportional to
also help to promote the protection of the pulp. Deposition the depth of cavity preparation. Luster [17] reported that
of hard tissue (reactionary dentin) is often observed in 28 resin monomers may affect the immune system and induce
days [14]. In the present study it was observed in groups DA immunosuppresion, resulting in decreased host resistance to
and DAC at 30 days. The presence of this tissue demon- infectious agents. Consequently, according to Jontell et al.
strates variable degrees of irritation promoted by the dentin [18] immunosuppression of pulpal immune competent cells
adhesive system tested, capable of causing synthesis as well elicited by resin components may enhance the potential for
as deposition of reactionary dentin matrix. bacterial injury to the pulp tissue. Thus, increased incidence
The persistent inflammatory reaction observed in one and severity of pulpal infection are justifiable concerns
specimen for group DA at 60 days showed that the pulp following exposure of the tissue to immunotoxic chemicals.
tissue cannot be repaired when resin components reach In addition, such as demonstrated by Gerzina and Hume
the connective tissue, as has been demonstrated when the [8] and according to Gwinnett and Tay [16], the present
same material was implanted into subcutaneous tissue of study showed that neither the hybrid layer nor a thin remain-
rats [15]. Gwinnett and Tay [16] showed a similar chronic ing dentin between the cavity floor and pulp tissue is totally
 J. Hebling et al. / Journal of Dentistry 27 (1999) 557–564 563

capable of preventing resin monomer diffusion across the the use of staining methods to evaluate the bacteria presence
dentinal tubules. Following dentin etching, faster outward has one more disadvantage. This method gives no informa-
dentinal fluid movement seems to interfere with complete tion about species, pathogenicity or viability of the bacteria
polymerization of fluid resin [11]. According to Hussey present.
[19], the heating caused during the light curing of composite According to the histological findings in the present study
resin reverses the dentinal fluid movement and, as conse- and those reported from relevant literature, we have been
quence, the inward fluid movement may carry the unpoly- able to determine that the RDT is an important factor to
merized resin particulates through dentinal tubules to reach protect the pulp tissue from the cytotoxic effects of the
the pulp tissue. Bouillaguet et al. [5] demonstrated the diffu- adhesive system tested. The persistent inflammatory pulp
sion of HEMA through dentinal tubules even against inter- response seems to occur when the dental cavities have a
nal pulp pressure. In addition, after local anesthesia RDT of less than 300 mm. No strong relationship between
including vasoconstrictor, such as was used in this present bacteria and intensity of pulp response could be found in the
study, pulp pressure is decreased [11] and the inward dent- present study. In this way, the application of biocompatible
inal fluid can be increased [20], making easier the diffusion material on cavity floor of deep cavities is important to
of noxious substances across the dentinal tissue, especially protect the pulpo-dentin complex against chemical irritation
substances of small molecular size. Based on our findings promoted by dental materials which can release unpolymer-
and reports in the literature we suggest that the monomers, ized monomeric globules and dissolved resin to diffuse
which reached the pulp tissue, were responsible for the through dentinal tubules.
persistent inflammatory reaction observed through the last
period of evaluation.
Acknowledgements
Special attention has been paid to bacterial presence at the
tooth-restoration interface and its possible role in irritation
The authors thank Dr. Carl Thomas Hanks, Department
of the pulp tissue. While a strong relationship between
of Oral Pathology-University of Michigan, for reviewing
bacterial presence and inflammatory pulp response has
the manuscript and for the wise suggestions and also
been reported [21], it could not be demonstrated in the
Pedro Carlos Oprime, Technological Center of information
present study. Pulp inflammation with absence of bacteria
in Materials-University of São Carlos (Brazil), for the statis-
as well as the reverse, i.e. large number of bacteria but no
tical analysis.
pulp inflammation was shown at all periods of evaluation.
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