SEM EVALUATION

J Bagh College Dentistry Vol. 26(1), March 2014 An in-vitro scan

Restorative Dentistry 42

An in-vitro scan electron microscope comparative study of

dentine-Biodentine interface

Jameel M. A. Sulaiman, B.Sc., M.Sc. (1)

Maha M. Yahya, B.D.S., M.Sc. (2)

Wiaam M.O. Al-Ashou, B.D.S., M.Sc. (2)

ABSTRACT
Background: This research was an in-vitro SEM comparative study of Dentine – Biodentine TM interface.

Materials and Methods: Sixty three freshly extracted human molars, Biodentine TM (Septodont, France), MTA (ProRoot,

Tulsa, Brazil), GIC (MediFil, Promedica, Germany), light microscope, scaler and pumice, high speed hand piece,

diamond bur, Scan Electron Microscope: VEGA\\ Easy Probe. TESCAN – Germany. The study was performed first at

the University of Mosul, College of Dentistry to dental models were brought the sixty-three of the specialty dental

health center in Mosul. The teeth was prepared by cleaning, cutting, and removing all the caries and examined

under light microscope and decayed teeth was excluded .Then the teeth was divided randomly into three main

groups (A, B, C) and each major group was divided into three sub groups: (A1, A2, A3) was filled with (Biodentine TM),

(B1, B2, B3) was filled with (MTA) and (C1, C2, C3) was filled with the (GIC). Each subset contains seven (7) samples. All

groups were filled according to the manufacturer instructions, and then restored at 37°C and 100% humidity. After

storage periods of (7, 14, 28) days, the teeth were sectioned mesio-distaly using a low speed diamond saw (Isomet,

Buehler Ltd.), and examined under SEM at the University of Technology-Nano Research Center in Baghdad.

Results: Under the condition of this in vitro study, examination with SEM showed that the marginal gaps between the

experimental materials and the dentine is time dependant, with the best results was observed between Biodentine

and dentine interface.

Conclusion: The marginal gaps between the experimental materials and the dentine are time dependent.

Keywords: Interface, Biodentine TM, MTA, SEM. (J Bagh Coll Dentistry 2014; 26(1):42-48).

الخالصة

: ثالثة المواد وطرائق العمللمادةعاج السن الطبيعي مع المادة الصناعية المثيلة لها. : يهدف البحث إلى استخدام تقنية المجهر االليكتروني إلجراء دراسة مقارنة للوسط البيني األهداف

:SEM)، المجهر االليكتروني الماسح (GIC)والـ (MTA)، ومادتي الـ TM (Biodentine(ـالاد مختلفة من الحشوات السنية ( عينة من األسنان الطبيعية ، ثالثة مو36وستون )

VEGA\\ Easy Probe. TESCAN - Germany) أسنان، مجهر ضوئي ، مقحلة(Scaler) ومسحوق(Pumice) أدوات باإلضافةإلىالماس، قاطع من، رأس األسنانجة لمعال
من المركز الصحي الـثالثة والستون نماذج األسنان بعد أن تم جلبفي جامعة الموصل كلية طب األسنان أوال أجريت الدراسة الرطوبة. ف من يدوية عالية السرعة، جهاز تجفي

األسنانالتخلص من لغرض جميع التسوسات وفحصها بالمجهر الضوئي وإزالةمل التنظيف والقص لتش للعمل البحثي تحضير العيناتبدأ العمل لثم التخصصي لألسنان في الموصل

(لمادة الـ A)A2,A1,3(ثانوية ثالثة مجاميع إلىوالمجموعة الرئيسية الواحدةA, B, C)(وهي ثالثة مجاميع رئيسية إلىعشوائيا األسنانقسمت. المنخورة
TM (Biodentine ،

)3,B2,B1(B لمادة الـ(MTA) و)3,C2,C1(C لمادة الـ(GIC) بالحشوات السنية المخصصة لهذه الدراسة ثم بدأت المعالجة . عينات (7)سبعة على تحتوي ثانوية كل مجموعة. و

ألماني من شركة –(GIC)الـ اما مادة ,ProRoot) (Tulsaبرازيلي من شركة (MTA)الـ ، ومادة(Septodont)فرنسي الصنع من شركة Biodentine) TM(وهي مادة الـ

(MediFil, Promedica) ،لص للتخ از تجفيفهبج جميع العينات ( لكل مجموعة ، ثم جففت28 ,14 ,7وهي ) باأليامترات زمنية مختلفة ولف بالماء المقطر ميلة الخزنبدأت ع بعدها
. في بغداد مركز بحوث النانوتكنولوجي –الجامعة التكنولوجية في (SEM)بجهاز المجهر االليكتروني الماسح ريلتصولأرسلت بعدهاوتم تقطيعها ثم من الرطوبة

وسطح TM (Biodentine( مادة الـالنتائج لوحظت بين أفضلمقدار الفجوة الحاصلة بين عاج السن والمواد المفحوصة تعتمد على الزمن وان نتائج هذه الدراسة أن أظهرتالنتائج:

عاج السن.
مرتبط بعامل الزمن. األسنانعاج السطحية بين المواد المفحوصة وسطحمقدار الفجوة إن :االستنتاجات

INTRODUCTION
Torbinejad first developed mineral trioxide

aggregate (MTA) as a surgical root repair material

in 1993 (1). Subsequently, significant interest has

been shown in MTA, due to its compatibility (2)

and potential bioactivity (3). More recently, a new

calcium-silicate restorative material called

Biodentine TM has been introduced by Septodent,

to be used not only as an endodontic repair

material but also as a coronal restorative material

for dentin replacement.

Biodentine TM consists of a powder and liquid

in apipette. The powder mainly contains

tricalcium and diecalcium silicate, the principle

component of Portland cement and MTA, as well

as calcium carbonate zirconium dioxide serves as

contrast medium (4).
(1)Lecturer. Department of Basic Sciences. College of Dentistry,
University of Mosul.

(2)Lecturer. Department of Conservative Dentistry. College of
Dentistry, University of Mosul.

The liquid consist of calcium chloride in an

aqueous solution with an admixture of modified

poly carboxylate. The powder is mixed with the

liquid in a capsule in a toturator for 30 seconds,

sets in about 12 to 16 minutes (5).

Biodentine TM can be used for the treatment of

root perforation or for the pulp floor, internal and

external resorption, apexification, retrograde root

canal obturation, pulpotomy, and also for

temporary sealing of cavities and cervical filling
(6).

Biodentine TM with Active Biosilicate

Technology announced by dental material

manufacturer Septodent in September of 2010,

and made available in January of 2011,

Biodentine TM is a calcium silicate based material

used for crown and root repair treatment, repair of

perforation or desorption’s, apexification and

root-end filling. The material has indications
similar to calcium silicate based materials e.g.

MTA, Septodent claimed that Biodentine TM is not

mutagenic (7) and that it can resist microleakage (8).

J Bagh College Dentistry Vol. 26(1), March 2014 An in-vitro scan

Restorative Dentistry 43

Biodentine TM shares both its indications and

mode of powder in capsule and liquid in a pipette.

The powder mainly contains tri calcium and

dicalcium silicate, the principle component of

Portland cement as well as calcium carbonate,

zirconium dioxide serves as a contrast medium (9).

The liquid consists of calcium chloride in aqueous

solution with an admixture of polycarboxylate.

The powder is mixed with the liquid in a capsule

in the triturate for 30 seconds. Once mixed

Biodentine TM sets in about 12 minutes. During

the setting of cement calcium hydroxide is

formed. The consistency of Biodentine TM reminds

of that of phosphate cement (10, 11).

The aim of this present study is to investigate

the marginal interfaces created between

Biodentine TM, MTA, GIC and Dentine. The

sealing ability of these materials is assessed in-

vitro through SEM observation of the tooth-

cement interface.

MATERIALS AND METHODS
Sixty three freshly extracted human molars

were used for this study. After visual inspection

with a light microscope to ensure that the teeth did

not show any caries or cracks, the teeth were

cleaned and polished with scaler and pumice. One

standardized class I cavity in the occlusal surface

were prepared on each tooth. All manipulations

and restorations were performed by a single

experienced operator to prevent variations due to

operator’s skill. Cavities were prepared with a

high speed handpiece, using a diamond bur under

heavy water spray. The diamond bur was replaced

after every four preparations.

All internal line angles were rounded. The

overall dimensions and depths of cavities were

standardized as follows: occlusal floor width

4mm, length 5mm, depth 2.5mm. The occlusal

floor ended in dentine, just below the dentino-

enamel junction. The teeth were immediately and

randomly divided into nine groups (7 teeth for

each) according to the filling material used for the

restoration of the occlusal cavities and the time of

storage as follow:

Group A: filled with Biodentine and subdivided

into three sub groups (A1, A2, A3) with seven teeth

for each.

A1: Restored with Biodentine and stored for 7

days

A2: Restored with Biodentine and stored for 14

days

A3: Restored with Biodentine and stored for 28

days.

Group B: filled with MTA and subdivided into
three groups (B1, B2,B3) with seven teeth for each

subgroup.

B1: Restored with MTA and stored for 7 days

B2: Restored with MTA and stored for 14

days.

B3: Restored with MTA and stored for 28

days.

Group C: filled with Glass Ionomer cement and

subdivided into three groups (C1, C2, C3) with

seven teeth for each subgroup.

C1: Restored with Glass Ionomer cement and

stored for 7 days.

C2: Restored with Glass Ionomer cement and

stored for 14 days.

C3: Restored with Glass Ionomer cement and

stored for 28 days.

All groups were filled according to the

manufacturer instructions, and then restored at 37

°C and 100% humidity.

After storage periods, the teeth were sectioned

mesio-distally using a low speed diamond saw

(Isomet, Buehler Ltd.), thus passing through the

center of the restoration. Then the sectioned

specimens were cleaned with 10%

orthophosphoric acid (H3SO4) for 3 to 5 seconds

and quickly rinsed with air water spray for 15

seconds to remove the smear layer. Later all the

specimens were dehydrated by increasing

concentration of ethyl alcohol [C2H5OH] (30%,

50%, 70, 90% and 100%).

Once the specimens were dehydrated with

various concentrations of alcohol, they were

mounted with silver paste on metallic stubs and

gold coated with sputtering system under vacuum

desiccation and then examined under SEM

(VEGA Easy Probe – Germany),at acceleration

voltage of 10 to 30 KV.

The internal gaps between the dentinal surface

and dentine substitute materials were observed

under scanning electron microscope.

Representation photomicrographs were taken

at a magnification power of (1000-1200) X. The

internal gaps at different levels were measured in

each photomicrograph and mean was taken. The

values obtained in microns and the data were

calculated and analyzed statistically using

ANOVA and Duncan’s multiple range test at

(p<0.05).

RESULTS
Under the condition of this in vitro study,

examination with SEM showed that the interface

between group A (Biodentine TM) and human

dentin were approximately in intimate contact

after 28 days of storage (i.e. the gap observation

was 1µm). While during the first week the mean
diameter of the gaps between Biodentine TM and

the tooth structure was (8.1± 0.888 µm) and the

J Bagh College Dentistry Vol. 26(1), March 2014 An in-vitro scan

Restorative Dentistry 44

interface became more intimate after two weeks,

the mean diameter of the gaps was (3.16 ±

0.7638µm). And the difference between

Biodentine TM groups at different time was

statistically highly significant (p<0.01) as seen in

Figure (1), Table (1).

The result of this in vitro study showed that in

group B (MTA) the mean diameter of the gaps

was (54.467 ± 4.313 µm), (6.0 ± 1.0 µm) and

(3.333 ± 1.528 µm) was statistically highly

significant (p<0.01) as seen in Figure (1), Table

(2), for subgroup (B1,B2 and B3) respectively.

Group B3represent the lowest mean of gaps which

was not significantly differenced from groups B2
(P>0.05). Group B1 showed the highest mean of

the gaps, and the difference was significant when

compared with the group B2 and B3 (P<0.05) as

seen in Figure (1), Table (2).

The results also showed that in group C (Glass

Ionomer) the mean diameter of the gaps was

(7.27 ± 0.86 µm), (25.0 ± 6.26µm) and (64.0 ±

33.81µm) for subgroup (C1, C2 and C3)

respectively, and the difference was statistically

significant between these groups( P<0.05)as seen

in Figure (1), Table (3).

Duncan's multiple range test table(4) showed

that at 7 days storage period Glass Ionomer

Group represent the lowest mean of the gaps

(7.267 ± 0.862) µm which was significantly

different(P<0.05)when compared with Biodentine
TM group (8.1 ± 0.889µm) and MTA group(

54.467 ± 4.313µm) ,and difference was not

statistically significant between Biodentine TM

and MTA(P>0.05).

Duncan's multiple range test table (5) showed

that at 14 days storage period Biodentine TM

Group represent the lowest mean of the gap

(3.167 ± 0.764 µm) which was not significantly

different (P>0.05) when compared with MTA

group (6.0 ± 1.0 µm) and Glass Ionomer group

(25.0 ± 6.264 µm) which showed the highest

mean of gaps and the difference was highly

significant when compared with Biodentine TM

and MTA group (p<0.01).

Duncan's multiple range test table (6) showed

that at 28 days storage period Biodentine TM

Group represent the lowest mean of the gap (1.0 ±

0.000 µm) which was not significantly different

when compared with MTA group (3.33 ± 1.53

µm) and Glass Ionomer group (64.00 ± 33.81µm)

which showed the highest mean of gaps and the

difference was significant (p<0.05) when

compared with Biodentine TM and MTA group.

Duncan's multiple range test table (7), figure

(2), showed that at (7, 14, 28) days storage period

Groups (A, B, C) was highly significantly

different (p<0.01). There was no significant

between A1, A2, A3, B2, B3 and C1 (P<0.05), and

there was no significant between B1 and C3, but

there was a significant between C2 and the other

subgroups.

DISCUSSION
The quality and durability of the interface is a

key factor for the survival of a restorative material

in clinical conditions; the marginal adaption and

the intimate contact with the surrounding material

(dentine, enamel and dental material) are

determinative features (5,13). In the present study

this was investigated by scan electron microscope

(SEM) at magnification (1000-1200) X to assess

the interfacial seal between enamel and dentine

and three restorative materials (Biodentine TM,

GIC and MTA). SEM represents a valid tool for

evaluation of the marginal integrity in in-vitro

studies (14, 15). It is a widely used morphological

examination of different interface (16).

Additionally it is used to obtain a quantitative

evaluation of the extent of the marginal gaps (17-

19). Under the condition of this in-vitro study,

examination with SEM should that the interface

between Biodentine TM and human dentine are

approximately in intimate contact after 28 days of

storage (the gap was 1 µm) observer between

Biodentine TM and the tooth structure, while

during the first week the mean diameter of the gap

between Biodentine TM and the tooth structure was

(8.1 ± 0.888 µm) and the interface become more

intimate after two weeks, i.e. the mean diameter

of the gaps was (3.1667 ± 0.7638µm).

Santos et al (20) observed that the interfacial

gap of Biodentine TM - dentine may be compared

to the hard tissue layer shown to be formed when

using Pro-Root MTA which is considered as a

precipitation of Hydroxyapatite. Goldberg et al (21)

observed that SEM microphotograph showed the

occurrence of a cohesive failure with Biodentine
TM cement with alteration of the tooth-biomaterial

interfaces, hence providing evidence for the

quality of the micromechanical adhesion

occurring during the SEM preparation

Table (6) showed that there is a direct contact

(without a gap), between Biodentine TM and the

natural dentine. The cracks observed in side

Biodentine TM caused by dehydration due to SEM

sample preparation under vacuum (22). This

cohesive failure dose not affected the dentine –

Biodentine TM interface, which indicate the quality

of the micro-mechanical adhesion (23, 24).

In comparison of interface of Biodentine TM-

Dentine in tables (4), (5) and (6), the interfaces

were very similar in all of the subgroups (A1, A2,

A3), while in group (B) the mean diameter of the

gaps were gradually decreasing with time. The

interface between MTA and Dentine became more

J Bagh College Dentistry Vol. 26(1), March 2014 An in-vitro scan

Restorative Dentistry 45

intimate after (28) days of storage. The possible

reason for the decrease in diameter of gaps is the

slight expansion of MTA upon setting (25, 26). The

marginal adaptation of MTA has been assessed

using SEM (27, 28), the long term seal was measured

over a (12) weeks and (12) month period. These

studies reported good results with MTA; this may

be because of its moisture tolerance and long

setting time (29, 30).

In the present study, group C with GIC (C1,

C2, C3) showed a large gap between the GIC and

tooth structure, and this gap is increasing with

time. During setting, GIC absorb a considerable

amount of water, which may affect their sealing

ability and physical properties. Silica hydrogel

forming around the glass particles is likely to act

as a fluid reservoir. It also tends to undergo some

amount of shrinkage during the setting which can

cause loss of the marginal integrity (31,32). Glass

Ionomer (GIC) is a material with universal

properties as dentist substitute; its ability to

exhibit chemical bond to tooth structure provides

an excellent marginal seal. However the marginal

seal is compromised because of its dissolution in

tissue fluids and its technique sensitivity (33).

As conclusions; all of the studied materials

exhibited some degree of marginal gaps that are

time dependent. A positive correlation was found

between the marginal adaptation and time of

storage. Biodentine TM and MTA exhibited similar

performances that are better than GIC under

conditions of this study.

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Table 1: Duncan's Multiple Range Tests for difference in the gaps between the dentin and the

Biodentine TM at different time intervals.
Sub - Group Number Mean Std. Deviation Duncan's test

A1 7 8.1000 0.888 A

A2 7 3.1667 0.7638 B

A3 7 1.0000 0.0000 C

One-Way Analysis of Variance
Source DF SS MS F - test P - value

Factor 2 79.442 39.721

86.77 0.000 Error 6 2.747 0.458

Total 8 82.189

Table 2: Duncan’s Multiple Range Tests for difference in the gaps between the dentin and the

MTA at different time intervals.
Sub - Group Number Mean Std. Deviation Duncan's test

B1 7 54.467 4.313 A

B2 7 6.000 1.000 B

B3 7 3.333 1.528 B

One-Way Analysis of Variance
Source DF SS MS F P

Factor 2 4970.75 2485.37

339.89 0.000 Error 6 43.87 7.31

Total 8 5014.62

Table 3: Duncan’s Multiple Range Tests for difference in the gaps between the dentin and the

Biodentine TM at different time intervals.
Sub - Group Number Mean Std. Deviation Duncan's test

C1 7 7.27 0.86 A

C2 7 25.000 6.26 A

C3 7 64.000 33.81 B

One-Way Analysis of Variance
Source DF SS MS F P

Factor 2 5054 2527

6.41 0.032 Error 6 2366 394

Total 8 7420

http://www.researchgate.net/researcher/28648225_Buffy_Storm/
http://www.researchgate.net/researcher/13045870_Frederick_C_Eichmiller/
http://www.researchgate.net/researcher/39776434_Patricia_A_Tordik/
http://www.jendodon.com/article/S0099-2399(06)81004-6/abstract
http://www.jendodon.com/article/S0099-2399(06)81004-6/abstract
http://www.jendodon.com/article/S0099-2399(06)81987-4/abstract
http://www.jendodon.com/article/S0099-2399(06)81987-4/abstract
http://www.jendodon.com/article/S0099-2399(06)81987-4/abstract
http://www.jendodon.com/article/S0099-2399(06)81987-4/abstract
http://www.ingentaconnect.com/content/mksg/eos;jsessionid=44s89dtd822ai.alexandra
http://www.ingentaconnect.com/content/mksg/eos;jsessionid=44s89dtd822ai.alexandra

J Bagh College Dentistry Vol. 26(1), March 2014 An in-vitro scan

Restorative Dentistry 47

28- days - Storage 14- days - Storage 7- days - Storage
Group

Material

Sub-group (A3 ) Sub-group (A2 ) Sub-group (A1 )

Group -A

BiodentineTM

Sub-group (B3 ) Sub-group (B2 ) Sub-group (B1 )

Group - B

MTA

Sub-group (C3 ) Sub-group (C2) Sub-group (C1 )

Group - C

GIC

Fig. 1: SEM images at magnification (1000-1200) X for the interspaces gap between tested

materials and dentine

Table 4: Duncan's Multiple Range Tests for difference in the gaps between the Biodentine TM

and MTA and Glass Ionomer cement at 7 days.
Material Number Mean Std. Deviation Duncan's test

Biodentine TM 7 8.1000 0.889 A

MTA 7 54.467 4.313 B

Glass Ionomer 7 7.267 0.862 A

One-Way Analysis of Variance

Source DF SS MS F P

Factor 2 4378.40 2189.20

326.15 0.000 Error 6 40.27 6.71

Total 8 4418.68

J Bagh College Dentistry Vol. 26(1), March 2014 An in-vitro scan

Restorative Dentistry 48

Table 5: Duncan's Multiple Range Tests for difference in the gaps between the Biodentine TM

and MTA and Glass Ionomer cement at 14 days.
Material Number Mean Std. Deviation Duncan's test

Biodentine TM 7 3.167 0.764 A

MTA 7 6.000 1.000 A

Glass Ionomer 7 25.000 6.264 B

One-Way Analysis of Variance
Source DF SS MS F P

Factor 2 845.7 422.9

31.07 0.001 Error 6 81.6 13.6

Total 8 927.4

Table 6: Duncan's Multiple Range Tests for difference in the interface gap between the

Biodentine TM and MTA and Glass Ionomer cement at 28days
Material Number Mean Std. Deviation Duncan's test

Biodentine TM 7 1.00 0.00 A

MTA 7 3.33 1.53 A

Glass Ionomer Cement 7 64.00 33.81 B

One-Way Analysis of Variance
Source DF SS MS F P

Factor 2 7655 3827

10.03 0.012 Error 6 2291 382

Total 8 9946

Table 7: FOR ALL
Sub - Group Number Mean Std. Deviation Duncan's test

A1 7 8.1000 0.888 A

A2 7 3.1667 0.7638 A

A3 7 1.0000 0.0000 A

B1 7 54.467 4.313 C

B2 7 6.000 1.000 A

B3 7 3.333 1.528 A

C1 7 7.27 0.86 A

C2 7 25.000 6.26 B

C3 7 64.000 33.81 C

One-Way Analysis of Variance
Source DF SS MS F P

Factor 8 13693 1712

12.77 0.000 Error 18 2413 134

Total 26 16106

100

7 - Days
14 - Days

28 - DaysG
a

p
i

n
m

ic
ro

m
e

te
r

Time

Biodentine TM

MTA

GIC

Figure 2: Gap Distance with time storage for Biodentine TM, MTA and GIC.