COMPARISON OF THE POTENTIAL DISCOLOURATION

EFFECT OF MTA ANGELUS, ENDOCEM MTA AND NEO MTA

ON NATURAL TEETH – AN IN VITRO STUDY

Dissertation submitted to

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY

In partial fulfillment for the Degree of

MASTER OF DENTAL SURGERY

BRANCH IV

CONSERVATIVE DENTISTRY AND ENDODONTICS

MAY 2020

ACKNOWLEDGEMENT

I take this opportunity to sincerely thank my post graduate teacher and

my guide Dr. C. S. Karumaran, M.D.S., Professor, Department of

Conservative Dentistry and Endodontics, Ragas Dental College and Hospital,

for his patience, perseverance in motivating, guiding and supporting me

throughout my study period. His guidance, support, and constant

encouragement throughout my study period helped me to finish my thesis.

My sincere thanks to Dr. R. Anil Kumar, M.D.S., Professor and

HOD, Department of Conservative Dentistry and Endodontics, Ragas Dental

College and Hospital, who helped me with his guidance, during my study

period.

I extend my sincere thanks to Dr P. Shankar, M.D.S., Professor,

Ragas Dental College and Hospital, for his guidance and encouragement

during my study period.

I extend my sincere thanks to Dr. M. Rajasekaran, M.D.S.., Professor,

Ragas Dental College and Hospital, for his encouragement, motivation,

support and guidance all throughout my study period.

I extend my sincere thanks to Dr. B. Veni Ashok, M.D.S., Professor,

for his constant encouragement in academics and support throughout my

propaganda.

I would like to solemnly thank and be grateful to Dr. G Shankar

Narayan, M.D.S., Dr. S.M. Venkatesan, M.D.S., for their constant support,

guidance and encouragement throughout my propaganda.

I would like to thank Dr. B Venketesh, M.D.S., Dr. M. Sabari M.D.S,

Dr.Arrvind Vikram, M.D.S. Readers, for all their help and support during

my study period.

I would also like to thank Dr. C Nirmala, M.D.S., Dr. Shalini, M.D.S.,

Dr. V Sudhakar, M.D.S., Senior lecturers for their friendly guidance and

support.

I also wish to thank the management of Ragas Dental College and

Hospital, Chennai for their help and support.

I thank all my batchmates Dr. Anu Priya G, Dr. Anitha Varghese,

Dr. Akshaya V B, Dr. Vinaya Madhuri B, Dr. Gayathri, Dr. Sai Swathi R,

Dr, Suraj U, who had the patience to bear with me, my beloved seniors

especially Dr. Nandhini Devi, Dr. Darlene Ann Johnson and juniors

especially Dr. Azhagu Abirami, Dr. Roselin Stalin for their constant moral

support, guidance patience, love and encouragement during my period.

Nothing like a friend in need is a friend indeed, I’m blessed to

have my friends, Dr. Priyanka Venkatasubramaniam, Dr. Jayanthi,

Dr. Mahalakshmi, Dr. Vijendranath for all the help, support and

encouragement they have given me.

I would like to extend my gratitude to my father Fabian Sebastian, my

mother Judith Sebastian, my brother Benedict Roshan Sebastian my aunt

Maria O’ Connor and my philanthropist Stephen Dinesh for their constant

love, understanding, moral support and encouragement throughout these

years without which I would not have reached so far.

My sincere thanks to Mr.K.Thavamani for his patience and support

in DTP and Binding works. I extend my thanks to S.Venkatesan for his help

in statistical work.

Above all, I am thankful to God, who always guides me and has given

these wonderful people into my life.

LIST OF ABBREVIATIONS

SL.NO ABBREVIATIONS DESCRIPTION

1 MTA Mineral Trioxide Aggregate

2 GIC Glass Ionomer Cement

3 EDTA Ethylenediamine-tetraacetic acid

4 CEJ Cemento - enamel junction

5 SD

Standard Deviation

6 IBM.SPSS International Business Machines Statistical

Package for the Social Sciences

7 ANOVA Analysis of variance

CONTENTS

S. NO. INDEX PAGE.NO

1. INTRODUCTION 1

2. AIM AND OBJECTIVES 5

3. REVIEW OF LITERATURE 6

4. MATERIALS AND METHODS 23

5. RESULTS 30

6. DISCUSSION 37

7. SUMMARY 54

8. CONCLUSION 58

9. BIBLIOGRAPHY 61

10. ANNEXURES -

LIST OF TABLES

S.NO. TITLE

Table 1 MULTIPLE COMPARISONS OF THE GROUPS AT

DIFFERENT INTERVALS BY ONE-WAY ANOVA

Table 2 REPEATED MEASURES OF ANOVA FOR BLOOD ONLY

(∆E)

Table 3 PAIRWISE COMPARISONS OF BLOOD ONLY GROUP AT

VARIOUS INTERVALS

Table 4 REPEATED MEASURES OF ANOVA FOR MTA ANGELUS

(∆E)

Table 5 PAIRWISE COMPARISONS OF MTA ANGELUS GROUP AT

VARIOUS INTERVALS

Table 6 REPEATED MEASURES OF ANOVA FOR ENDOCEM MTA

(∆E)

Table 7 PAIRWISE COMPARISONS OF ENDOCEM MTA AT

VARIOUS INTERVALS

Table 8 REPEATED MEASURES OF ANOVA FOR NEO MTA (∆E)

Table 9 PAIRWISE COMPARISON NEO MTA GROUP AT VARIOUS

INTERVALS

LIST OF GRAPHS

S.NO. TITLE

Graph 1 REPRESENTING ∆E IMMEDIATE POSTOPERATIVE

VALUES OF THE GROUPS

Graph 2 REPRESENTING ∆E DAY 1 VALUES OF THE GROUPS

Graph 3 REPRESENTING ∆E 1 WEEK VALUES OF THE GROUPS

Graph 4 REPRESENTING ∆E 2 WEEKS VALUES OF THE GROUPS

Graph 5 REPRESENTING ∆E 3 WEEKS VALUES OF THE GROUPS

Graph 6 REPRESENTING ∆E VALUES OF BLOOD ONLY GROUP

AT VARIOUS INTERVALS

Graph 7 REPRESENTING ∆E VALUES OF MTA ANGELUS GROUP

AT VARIOUS INTERVALS

Graph 8 REPRESENTING ∆E VALUES OF ENDOCEM MTA

GROUP AT VARIOUS INTERVALS

Graph 9 REPRESENTING ∆E VALUES OF NEO MTA GROUP AT

VARIOUS INTERVALS

LIST OF FIGURES

S.NO. TITLE

FIGURE 1 EXTRACTED HUMAN UPPER CENTRAL INCISORS

FIGURE 2 DECORONATION OF TEETH AT CEJ

FIGURE 3 DIAMOND DISC

FIGURE 4 HANDPIECE

FIGURE 5 ISO DIAMOND CYLINDRICAL BUR – SR -13 (MANI)

FIGURE 6 SYRINGE (UNOLOK)

FIGURE 7 SALINE

FIGURE 8 SODIUM HYPOCHLORITE IRRIGATION SOLUTION 3%

(CHEMDENT)

FIGURE 9 DESMEAR 17% EDTA SOLUTION

FIGURE 10 MICROPIPETTE

FIGURE 11 MTA (MICRO ANGELUS)

FIGURE 12 ENDOCEM MTA (MARUCHI

FIGURE 13 NEO MTA PLUS (AVALON BIOMED)

FIGURE 14 GLASS IONOMER CEMENT TYPE IX (GC), MIXING

PAD AND AGATE SPATULA

FIGURE 15 GLASS SLAB AND CEMENT SPATULA

FIGURE 16 PLASTIC INSTRUMENT

FIGURE 17 CAVITY PREPARATION

FIGURE 18 SPECTROPHOTOMETER

FIGURE 19 MANIPULATION OF MTA ANGELUS

FIGURE 20 MANIPULATION OF ENDOCEM MTA

FIGURE 21 MANIPULATION OF NEO MTA

FIGURE 22 BLOOD ONLY GROUP

FIGURE 23 MTA ANGELUS GROUP

FIGURE 24 ENDOCEM MTA GROUP

FIGURE 25 NEO MTA GROUP

FIGURE 26 POST OPERATIVE SAMPLES

Introduction

Introduction

1

INTRODUCTION

Introduction of Mineral Trioxide Aggregate (MTA) as a root canal

filling material in the year 1993 by Torabinejad, has continued to remain as a

material of choice, widely reviewed and researched by the clinicians for

various properties. Currently it is the most preferred and the choice of material

for various clinical situations like pulpotomy, pulp capping, perforation

management, root canal fillings and regenerative endodontic procedures. 1

The basic composition of MTA is tricalcium silicate, tricalcium

aluminate, calcium silicate and tetracalcium aluminoferrite which is similar to

Portland cement. 2 The initially formulated GMTA(which is grey in colour)

had very few limitations and one among them is tooth discolouration when

used in the coronal aspect. 3

To overcome this drawback, the manufacturers introduced a newer

formulation called white MTA. The newer MTA has reduced Al2 O3, MgO

and FeO when compared with grey MTA. This reduction in FeO resulted in

less aluminoferrite phase which is the main constituent that is responsible for

grey colour in GMTA. Nevertheless GMTA and WMTA contributes to the

tooth discolouration. 1,2,4–8

MTA has a pH around 12.5 and the ideal setting environment is

alkaline. The setting time for grey and white MTA is 165 mins. 9 The

prolonged setting time of the cement 10

is not an ideal property when used as a

Introduction

2

root end filling material, as it is in contact with blood and tissues which

reduces the pH too. There is a drop in pH and results in washout of the

material. 11

This initiated in introducing newer cements with less setting time

which was brought about by adjusting the original composition of the

cement.12

The ideal property of the MTA cement is not achieved when there is

an interference with the setting time. When MTA is placed in contact with

blood, discolouration occurs by haemolysis of the erythrocytes.4 Also altered

pH influence the setting time and final set property of the MTA material.13

This is eliminated by newer introduction of MTA like MTA Angelus,

Endocem MTA and Neo MTA.

MTA Angelus (Angelus, Londrina, Brazil) was launched in Brazil in

2001 and received FDA approval in 2011, making it available in the United

States. MTA Angelus sets within 15 minutes of being prepared which is

desirable with clinicians without worrying about MTA washout. The reduced

setting time of MTA Angelus is due to the decreased concentration of calcium

sulfate, which is the substance responsible for the longer setting time in the

original formulation.

Due to the slow setting property of MTA [3-4 hours], calcium

silicate based fast setting Endocem MTA material has been developed.14

Endocem MTA is non-miscible with liquid components, including blood and

is ideal for use in clinical situations where bleeding is difficult to control.

Introduction

3

Endocem MTA employs the pozzolanic reaction, possessing cementitious

properties and known to reduce the setting time of Portland cement.14

Neo MTA Plus (Avalon Biomed Inc, Bradenton, FL), a new material

similar to MTA has been marketed for use in pulpotomies as it does not

discolour the tooth.15

MTA contains bismuth oxide which has been reported to

discolour, this is replaced by tantalum oxide in Neo MTA. These radiopacifers

were inert and showed no interference with the hydration of the material

without phase changes and did not result in discolouration.15

Discolouration caused by MTA is exacerbated by its contact with

blood.4

Most theories are related to:

The presence of bismuth oxide in the WMTA formulation which

causes the discolouration.16

Collagen in dentin matrix, reacted with bismuth oxide, resulting in

greyish discolouration.17

The release of heavy metal ions from MTA is also reported to cause

discolouration.

The slow setting reaction of MTA permits the absorption and

haemolysis of erythrocytes, resulting in material and subsequent tooth

discolouration when in contact with blood.4

Introduction

4

This in vitro study was done to compare the potential discolouration

effect of MTA Angelus, Endocem and Neo MTA since only few studies have

been done on the discolouration potential.

Aim and Objectives

Aim and Objectives

5

AIM AND OBJECTIVES

AIM:

The aim of this study is to compare the potential discoloration effect of

MTA Angelus, Endocem and Neo MTA on natural teeth.

OBJECTIVES:

1. This study is designed to evaluate the discoloration potential of

MTA when placed in contact with blood.

2. To compare the discoloration potential among MTA Angelus, Endocem

and Neo MTA when placed in contact with blood using a

Spectrophotometer.

Review of Literature

Review of Literature

6

REVIEW OF LITERATURE

Van der Burgt and Plasschaert et al (1985)18

investigated the

staining potential of different materials like Cavit, Durelon, Dycal, Fletcher’s

cement, IRM, AH26-silver free, gutta-percha, Duo Percha, Fuji ionomer. and

zinc phosphate cement by means of a visual method. The standards were

arranged according to hue, value, and chroma as recommended by Munsell.

Seghi et al (1990)19

evaluated the effects of instrument-measuring

geometry on color-difference assessments made on dental porcelains. The

results indicate that a high degree of correlation can exist between color-

difference measurements regardless of the design of the instrument-measuring

geometry. This work suggests that the development of clinically useful

devices need not be restricted to more traditional integrating-sphere-type

designs and that more photometrically efficient alternative designs should be

explored.

Mahmoud Torabinejad et al (1994)13

investigated mineral trioxide

aggregate (MTA), as a potential alternative restorative material to the

presently used materials in endodontics. Several in vitro and in vivo studies

have shown that MTA prevents microleakage, is biocompatible, and promotes

regeneration of the original tissues when it is placed in contact with the dental

pulp or periradicular tissues. He described the clinical procedures for

Review of Literature

7

application of MTA in capping of pulps with reversible pulpitis, apexification,

repair of root perforations nonsurgically and surgically, as well as its use as a

root-end filling material.

Thomas R. Pitt Ford et al (1995)20

investigated the histologic

response to intentional perforation in the furcations of mandibular premolars

in seven dogs. In half the teeth, the perforations were repaired immediately

with either amalgam or mineral trioxide aggregate; in the rest the perforations

were left open to salivary contamination before repair. Results showed that the

mineral trioxide aggregate is a far more suitable material than amalgam for

perforation repair, particularly when used immediately after perforation.

Marin et al (1997)21

investigated tooth staining following pulpal

haemorrhage. Samples of whole blood, erythrocytes, plasma and platelet

concentrate and saline were individually placed in the pulp chambers of

groups of five teeth and centrifuged twice daily for 25 min over a period of 3

consecutive days. He confirmed that the blood pigment responsible for the

staining was found only in those samples containing erythrocytes. The tests

done by Marin et al showed that, following haemolysis of erythrocytes within

dentine, haemoglobin was found either intact or as one of the haematin

molecules with no further breakdown of the haem structure and no evidence of

any free ferric ions or haemosiderin.

Review of Literature

8

D.C.N. Chan et al (1999)22

studied the radiopacity of tantalum oxide

in filled resins at varying percentage loadings. Ta2O5 nanoparticles were

dissolved in methanol or powder forms were mixed into either glycerol

dimethacrylate (GDMA) or a bisGMA and specimens were made in a split

brass mold and compared with an aluminum step wedge (99.5% pure Al) and

a dentin slice of the same thickness. The radiopacity increased significantly

with tantalum loading and reached that of 70% enamel opacity.

Moir et al (2003)23

describes the nature of Portland (calcium

silicate-based) cements and outlines the manufacturing process and the quality

control procedures employed. Portland cement is essentially calcium silicate

cement, which is produced by firing to partial fusion, at a temperature of

approximately 1500°C, a well-homogenized and finely ground mixture of

limestone or chalk (calcium carbonate) and an appropriate quantity of clay or

shale. The composition is commonly finetuned by the addition of sand and/or

iron oxide. Cement making is essentially a chemical process industry and has

much in common with the manufacture of so-called heavy chemicals, such as

sodium hydroxide and calcium chloride. Close control of the chemistry of the

product is essential if cement with consistent properties is to be produced. This

control applies not only to the principal oxides which are present but also to

impurities, which can have a marked influence on both the manufacturing

process and cement properties.

Review of Literature

9

Asgary et al (2004)24

compared the composition of white mineral

trioxide aggregate (WMTA) and two different white Portland cements

(WPCs). The samples were prepared and then imaged in a JEOL JSM6400

scanning electron microscope, equipped with an Oxford Instruments light

element energy dispersive spectrometer detector for determining the elemental

composition. The electron probe microanalysis showed that lime (CaO) and

silica (SiO) were the dominant compounds in each case but with no detectable

trace of bismuth oxide (BiO) in WPCs. He concluded that, there is no

significant difference between the dominant compounds in both WMTA and

WPCs except the presence of bismuth oxide in WMTA.

L. Turanli et al (2004)25

studied the effect of three different natural

pozzolans from Turkish deposits on the properties of blended cements

produced by intergrinding cement clinker with a high volume of natural

pozzolan (55 wt.% of the cementitious material). The particle size distribution

of blended cements, setting time, heat of hydration, and compressive strength

of blended cement mortars were determined. Experimental results showed that

the hardness of the pozzolanic material strongly influenced the particle size

distribution and the related properties of the blended cements by affecting the

fineness of the components of the blended product. The early strength of the

mortars was strongly affected by the particle size distribution of blended

cements, whereas the strength development performance of the mortars was

Review of Literature

10

more related to the pozzolanic activity of the natural pozzolan present in the

blended cement.

Ahmed Al-Kahtani et al (2005)26

evaluated the seal created by

varying depths of mineral trioxide aggregate (MTA) plugs placed in an

orthograde fashion in five groups of 10 teeth. One group received a 2 mm

thick orthograde apical plug of MTA, the second group a 5 mm apical MTA

plug, and the third group a 2 mm apical MTA plug with a second 2 mm

increment, 24 h later. The remaining portion of the canal in these groups was

left unfilled. Group four received a 2 mm MTA plug that set for 24 h and the

canal was then back-filled with gutta percha and eugenol based sealer. Group

five was a positive control without an MTA plug. The apical seal was tested

using a bacterial leakage model of Actinomyces viscosus. Results showed a

statistically significant difference in only the 5 mm apical plug, which

completely prevented bacterial leakage.

W. T. Felippe et al (2006)27

evaluated the influence of mineral

trioxide aggregate (MTA) on apexification and periapical healing of teeth in

dogs with incomplete root formation and previously contaminated canals and

to verify the necessity of employing calcium hydroxide paste before using

MTA. He concluded that Mineral trioxide aggregate used after root canal

preparation favoured the occurrence of the apexification and periapical

healing. The initial use of calcium hydroxide paste was not necessary for

Review of Literature

11

apexification to occur, and has shown to be strongly related to the extrusion of

MTA and formation of barriers beyond the limits of the root canal walls.

Iwamoto et al (2006)28

evaluated teeth clinically and histologically

using white proroot MTA in direct pulp capping. white MTA were diagnosed

as clinically successful, i.e. an absence of clinical symptoms and did not show

evidence of periapical pathosis. Histologically dentin bridge had developed.

Iwamoto et al concluded that white ProRoot MTA was equally successful as

calcium hydroxide when used for direct pulp capping in mechanically exposed

teeth.

Song et al (2006)29

observed that difference between white and gray

MTA was the lack of iron ions in white MTA. the principal components of the

gray-colored formula are tricalcium silicate, bismuth oxide, dicalcium silicate,

tricalcium aluminate, tetracalcium aluminoferrite, and calcium sulfate

dehydrate, and the white-colored formula lacks the tetracalcium

aluminoferrite. The fluxing agent is used for production of the white version to

remove the ferrite phase during the clinkering process. Portland cement

differed from MTA by the absence of bismuth ions and presence of potassium

ions.

Bozeman et al (2006)30

stated that crystal growth and elemental

dissolution characteristics of gray Mineral Trioxide Aggregate (GMTA), white

MTA (WMTA), and an experimental material, Dentalcrete, were compared.

Review of Literature

12

For part A, comparing amount and composition of surface crystal growth,

twelve cylinders of each material were suspended in Phosphate Buffered

Saline (PBS) solution without Ca. The crystals were analyzed by Scanning

Electron Microscopy (SEM), X-ray Diffraction (XRD), and Inductively

Coupled Plasma—Atomic Emission Spectroscopy (ICP-AES). For part B,

three cylinders of each material were suspended in distilled, deionized water.

The water was analyzed by ICP-AES for Ca content at 24 h, 72 h, and 5, 7, 10,

and 14 days. Data were analyzed using one-way ANOVA and Tukey test.

Both MTA materials released more Ca initially, followed by a decline and

then rise in elution. GMTA produced the most surface crystal, which may be

clinically significant. The crystals on GMTA and WMTA were chemically

and structurally similar to hydroxyapatite (HA).

Lindsey et al (2007)31

determined the perceptibility and acceptability

of tooth color differences using computer-generated pairs of teeth with

simulated gingival displayed on a calibrated color monitor using appropriate

signal detection theory methodology. Responses to tooth color differences

(DE) were measured on each of the three principal axes of CIELAB color

space (L*, a*, and b*). No group differences among subjects were found. All

gave 50% match or acceptance points that averaged about 1.0 DE units in the

L* and a* directions, and 2.6 units in the b* direction.

Kim-Pusateri et al (2009)32

evaluated the reliability and accuracy of 4

dental shade-matching instruments in a standardized environment. Four shade-

Review of Literature

13

matching devices were tested: SpectroShade, ShadeVision, VITA Easyshade,

and ShadeScan. Color measurements were made of 3 commercial shade

guides (Vitapan Classical, Vitapan 3D-Master, and Chromascop). Shade tabs

were placed in the middle of a gingival matrix (Shofu GUMY) with shade tabs

of the same nominal shade from additional shade guides placed on both sides.

Measurements were made of the central region of the shade tab positioned

inside a black box. For the reliability assessment, each shade tab from each of

the 3 shade guide types was measured 10 times. For the accuracy assessment,

each shade tab from 10 guides of each of the 3 types evaluated was measured

once. Accuracy of devices was as follows: VITA Easyshade - 92.6%;

ShadeVision - 84.8%; SpectroShade - 80.2%; and ShadeScan - 66.8%.

Schembri et al (2010)33

studiedPortland cement with a four to one

addition of bismuth oxide marketed as mineral trioxide aggregate (MTA),

which is used mainly as a dental material.Measurements of arsenic, lead, and

chromium in hydrated gray and white Portland cement, ProRoot MTA, and

MTA Angelus were conducted with graphite furnace atomic absorption

spectrophotometry becauseheavy metal inclusion was a concern as MTA is in

contact with hard and soft tissues. It was concluded that both MTAs released

more arsenic than the amount specified. Portland cements and MTAs showed

evidence of heavy metals in the acid-soluble form as well as leaching in

deionized water and SBF. MTA contained levels of arsenic higher than the

safe limit specified.

Review of Literature

14

Ilya Belobrov et al (2011)34

describes the treatment of tooth

discoloration caused by white MTA used for the management of a

complicated crown fracture. A partial pulpotomy was performed with the use

of WMTA after a complicated crown fracture of the upper right central

incisor. Seventeen months later, upon access, the WMTA was completely

discolored. the WMTA was removed because of tooth discoloration, and

internal bleaching was performed. The toothremained vital, and adentin

bridge was confirmed clinically and radiographically. He Concluded that

WMTA used for vital pulp therapy in the esthetic zone may need to be

reconsidered.

Daniel Felman et al (2013)4 characterized discoloration when white

MTA was placed in the coronal aspect of the root canal ex vivo and the

influence of red blood cells on this discoloration. Color was assessed using

standardized digital photographs. All teeth discolored when restored with

wMTA, which was most prominent in the cervical third of the crown. The

presence of blood within the canal adjacent to the setting wMTA exacerbated

the discoloration.

Ioannidis et al (2013)6 evaluated specific alterations in tooth colour

with white and grey MTA when used to fill pulp chambers.Forty-five fully

developed, intact, mandibular third molars were sectioned 1 mm below their

cemento-enamel junction (CEJ). Their pulp chambers were chemo-

mechanically debrided, and the specimens were randomly assigned into three

Review of Literature

15

groups: Group 1–white MTAAngelus, Group 2– grey MTAAngelus and

Group 3–negative control (unfilled). During the experimental period,

specimens were immersed in vials containing distilled water up to the CEJ.

The discoloration was measured with a spectrophotometer and the data were

transformed into values of the CIE L*a*b* colour system and ∆E values were

obtained. He concluded that application of both grey and white MTA

formulations induced a decrease in lightness and reduction in redness and

yellowness in teeth. The present findings suggest that application of grey

MTA in the aesthetic zone should be avoided, whilst white MTA should be

used with caution when filling pulp chambers with the materials.

Ji-Hyun Jang et al (2013)35

evaluated tooth discoloration after the use

of mineral trioxide aggregate (MTA) and to examine the effect of internal

bleaching on discoloration associated with MTA. He observed that the

ProRoot and Angelus groups displayed increasing discoloration during a

period of 12 weeks. The discoloration associated with ProRoot and Angelus

was observed at the MTA-dentin interface and on the interior surface of the

dentin. He concluded then ProRoot and Angelus caused tooth discoloration.

However, Endocem did not affect the contacting dentin surface. Removing the

discolored MTA materials contributed more to resolving the tooth

discoloration than post-treatment internal bleaching.

Marta Valles et al (2013)16

used five different calcium silicate

cements namely ProRoot WMTA, Angelus WMTA, White Portland Cement

Review of Literature

16

[PC], PC with bismuth oxide, and Biodentine. They exposed each group of

cements to combined environment of light and anaerobic condition and

evaluated the color change in each specimens in different time period through

spectrophotometer. From the results he concluded Biodentine and Portland

cement demonstrated color stability than the other cements such as ProRoot

WMTA, Angelus WMTA, White Portland with bismuth oxide.

Marina Angélica Marciano et al (2014)17

studied if the increase in

radiopacity provided by bismuth oxide is related to the color alteration of

calcium silicate-based cement. Calcium silicate cement was mixed with 0%,

15%, 20%, 30% and 50% of bismuth oxide (BO), determined by weight and

Mineral trioxide aggregate was the control group. The assessments were

performed using a spectrophotometer to obtain the ΔE, Δa, Δb and ΔL values.

He concluded that increase in radiopacity provided by bismuth oxide has no

relation to the color alteration of calcium silicate-based cements.

Todd Berger et al (2014)36

investigated the role of bismuth oxide, a

constituent of contemporary mineral trioxide aggregate (MTA) materials,

andits response to various solutions that may contribute to the potential

discoloration. He observed thatall forms of ProRoot MTA showed

discoloration and concluded that exposing MTA in various forms to a variety

of liquids has determined that bismuth oxide in combination with other

chemical moieties is the prime cause of staining.

Review of Literature

17

Josette Camilleri et al (2014)37

viewed that Immersion of white MTA

and bismuthoxide in sodium hypochlorite resulted in the formation of a dark

brown discoloration. This change was not observed in Portland cement. He

concluded that Contact of white MTA and other bismuth-containing materials

with sodium hypochlorite solution should be avoided.

Hannah Beatty et al (2015)38

compared tooth discoloration between

ProRootMTA, Biodentine, and EndoSequenceRoot Repair Material. She used

bovine mandibular incisors and prepared them from the apical aspect after root

resection. Canals were prepared with sequentially larger ParaPost drills

coronal to the cementoenamel junction. Experimental materials were

condensed into the crowns and the access sealed. Color was assessed at

various times up to 2 months according to the CIE L*a*b* color space system

and concluded that BioDentine and EndoSequence root repair material

discolor bovine tooth structure to a perceptible degree. At 8 weeks, this was

significantly more than ProRootMTA.

Josette Camilleri et al (2015)15

evaluated three materials namely Neo

MTA Plus (Avalon Biomed Inc, Bradenton, FL), MTA Plus(Avalon Biomed

Inc), and Biodentine (Septodont, Saint-Maur-des-Foss_es, France) that are

used for pulpotomy procedures in immature permanent teeth to view their

color stability in the presence of sodium hypochlorite. He compared the color

stability using photography, spectrophotometry, and X-ray diffraction

analysis. He concluded all materials used in the study are suitable to be used

Review of Literature

18

pulpotomy procedure of immature teeth as all the material produced calcium

hydroxide as their byproduct in their early stage.but when comparing color

stability Neo MTA Plus and Biodentine are suitable alternatives to MTA, and

they do not exhibit discoloration.

BehnazEsmaeili et al (2015)39

compared the discoloration potential of

calcium enriched mixture cement, white mineral trioxide aggregate and

calcium hydroxide, after placement in pulp chamber. The highest ΔE value

belonged to WMTA group. They concluded that CEM cement may be the

material of choice in the esthetic region, specifically pertaining to its lower

colorchanging potential compared to WMTA.

Shin-Hong Kang et al (2015)3 compared the discolorationof these

various MTA-based materials and concluded less discoloration was observed

with ENDOCEM Zr and RetroMTA which contain zirconium oxide than with

ProRoot MTA and MTA Angelus which contain bismuth oxide

ZohrehKhalilaket al (2015)40

The aim of this in vitrostudy was to

compare discoloration induced by tooth colored mineral trioxide aggregate

(MTA) and calcium-enriched mixture (CEM) cement in extracted human

teeth. Color measurement was carried out by spectrophotometry and observed

that tooth discoloration was similarly detectable with both of the two

experimental materials.

Review of Literature

19

Marta Valles et al (2015)41

assessed the color stability of teeth

restored coronally with WMTA or Biodentine under artificial light. In this

invitro study he prepared cavities on coronal tooth and restored with WMTA +

composite, Biodentine + composite, or composite alone. Color was assessed

spectrophotometrically at 6 time points (initial, 1 week, 2 weeks, 1 month, 3

months, and 6 months), and color difference values were calculated. He

concluded teeth treated with WMTA exhibited discoloration, whereas those

treated with Biodentine maintained color stability throughout the study.

Christian A. Dettwiler et al (2016)42

viewed to investigate the

discoloration potential of different endodontic cements, dressings, and

irrigants used in dental traumatology. The specimens were selected and the

cavities were filled with a range of endodontic materials,sealed with

composite and stored in physiological saline. The color of the labial enamel

surface was measured with a spectrophotometer at 7 time intervals. After 12

months, significant staining was observed among the endodontic cements only

in the Portland cement group with additional bismuth oxide and other

commercially available calcium silicate cements containing bismuth oxide

were not significantly discolored. He concluded that the presence of bismuth

oxide in calcium silicate cements was not shown to be a reliable predictor for

tooth discoloration.

Review of Literature

20

Noushin Shokouhinejad et al (2016)43

compared the discoloration

potential of MTA namely ProRoot MTA (Dentsply Tulsa Dental Products,

Tulsa, OK) and calcium silicate based material such as Biodentine (Septodont,

Saint Maur des Foss_es, France), OrthoMTA (BioMTA, Seoul, Korea), and

EndoSequence Root Repair Material (ERRM; Brasseler, Savannah, GA) in the

presence or absence of blood. He concluded all materials showed discoloration

in the presence of blood butbiodentine and ERRM showed significantly less

tooth discoloration in the absence of blood.

Yoldas et al (2016)1 evaluated and compared the discoloration

potential of 3 different tricalcium cements using a bovine tooth model. He

used 4 groups namely BioAggregate, Biodentine, mineral trioxide aggregate

Angelus, and only blood.Crowns separated from the roots and materials have

been placed to the standardized cavities on the lingual surfaces of the crowns,

and their contact with blood has been provided. The color values of the

samples were measured with a digital tooth shade before the placement of the

materials, after the placement of the materials, in the 24th hour, in the first

week, in the first month, in the third month, and in the first year. Results

showed all groups displayed increasing discoloration during a period of the

first year. The ‘‘only blood group’’ showed the highest color change values,

and it was followed as Bio-Aggregate, mineral trioxide aggregate Angelus,

and Biodentine, respectively. He finally concluded that Biodentine is found to

have the least discoloration potential among the tested materials.

Review of Literature

21

Marina Angelica Marciano et al (2017)44

investigated the addition of

variable amounts of zinc oxide to inhibitdental discoloration caused by

mineral trioxide aggregate.He used MTA Angelus andMTA with additions of

5%, 15%, and 45% zinc oxide in weight were tested the set cements using a

combination of scanning electronmicroscopy, energy dispersive spectroscopy,

and x-raydiffraction. The pH and calcium ion releasewere measured after 3

hours, 24 hours, and 28 days.Dental discoloration in contact with the cements

wasmeasured after 24 hours, 28 days, and 90 days. The results showed

addition ofZnO did not alter significantly the radiopacity, settingtime, volume

change, pH, and biocompatibility compared with MTA Angelus. He

concluded the addition of 5%, 15%, or 45% zinc oxide to MTAAngelus

inhibits dental discoloration without modifyingthe radiopacity, setting time,

volume change, pH, andbiocompatibility.

Bansode et al (2018)45

reviewed tricalcium silicate based cements like

MTA though initially introduced as root end filling materials, have found wide

acceptance in other treatment modalities like pulp capping, pulpotomies,

apexification, perforation repairs because of their biocompatibility, sealing

abilities etc. At the same time its limitations like longer setting time, difficult

handling characteristics, potential to cause tooth discoloration have led to the

exploration of modifications of the material. This article reviews a newer

modification ‘Pozzolan Dental Cement’ which has been evaluated and

compared with the existing tricalcium silicate-based cement.

Review of Literature

22

Ivan Odler et al (2019)46

studied the hydration, setting and hardening

of Portland cement. There are various factors that determine the mechanism of

PC setting reaction. He has studied the chemistry in depth and given and

overview of the cement.

Jiménez-Sánchez et al (2019)47

studied the physicochemical

parameters with those of ProRoot MTA White (Pro) and NeoMTA Plus.

Setting time was assessed according using a scanning electron microscope.

Bioactivity evaluation in vitro was carried out, by soaking processed cement

disk in simulated body fluid (SBF) during 168 h. HP showed shorter initial

setting time compared to Pro and Neo and produce a quick and effective

bioactive response in vitro in terms of phosphate phase surface coating

formation which improved the outcome of vital pulp therapy.

Materials and Method

23

MATERIALS AND METHODS

ARMAMENTARIUM AND MATERIALS

1. 40 Extracted Human Upper Central Incisors

2. Diamond disc

3. Handpiece

4. ISO Diamond Cylindrical Bur – SR -13 (Mani)

5. Saline

6. Syringe (Unolok)

7. Sodium hypochlorite irrigation solution 3% (Chemdent)

8. 17% EDTA solution (Desmear)

9. Human Blood

10. Micropipette

11. MTA Angelus (Angelus)

12. Endocem MTA (Maruchi)

13. Neo MTA plus (Avalon Biomed)

14. Glass ionomer cement Type IX (GC)

24

15. Mixing pad and Agate spatula

16. Glass slab and Cement spatula

17. Plastic instrument

18. Spectrophotometer (Vita Easyshade Compact)

25

FLOW CHART ILLUSTRATING THE METHODOLOGY OF THE

STUDY

All the 40 teeth’s root were removed and crowns were

retained for the study.

A 3mm depth cavity was prepared with a cylindrical bur

(SR-13) on the palatal surface of the teeth.

40 extracted upper central incisors were collected.

The teeth were cleaned with pumice, rinsed and dried.

The teeth are then place in 3% sodium hypochlorite

solution for 30 mins, dried and placed in 17% EDTA

solution for another 2 mins for smear layer removal.

The teeth are divided into 4 groups

26

4 GROUPS

BLOOD ONLY

n=10

BLOOD + MTA ANGELUS

n=10

BLOOD + ENDOCEM MTA

n=10

BLOOD + NEO MTA

n=10

The access cavity of all the teeth were restored with Type IX GIC.

A preoperative shade was taken before placement of the respective

materials and an immediate postoperative shade was taken using a

spectrophotometer.

The shade was checked after 1 day

The shade was checked after 1 week

The shade was checked after 2 weeks

The shade was checked after 3 weeks

27

SELECTION AND PREPARATION OF THE SAMPLES:

BLOOD COLLECTION:

5ml blood was collected by venipuncture from a volunteer in an

anticoagulant spray coated tube that were sterile.

PROCEDURE:

40 extracted central incisors were collected. The teeth were cleaned

with pumice, rinsed with running water and dried. The crowns were separated

from their roots with a diamond disc. The crowns were retained for the study.

A cylindrical bur was used to prepare a cavity on the lingual surface of the

teeth of 3mm depth. The teeth were then rinsed and placed in 3% sodium

hypochlorite solution for 30 mins followed by 17% EDTA solution for 2 mins

for smear layer removal. The teeth were then rinsed and randomly divided into

4 groups:

Group A - Blood only;

Group B – Blood + MTA Angelus;

Group C – Blood + Endocem MTA;

Group D – Blood + Neo MTA

An initial shade was obtained using a spectrophotometer for all the

teeth. Each tooth was filled with blood using a micropipette, followed by their

respective group material and their access cavity were restored with

28

Type IX GIC. In Group A, the cavities were filled only with 2.0μL blood

followed by GIC restoration. In Group B, the cavities were filled with 0.5μL

blood followed by MTA Angelus using a plastic instrument and restored with

GIC. MTA Angelus was mixed as per the manufacturer’s instructions and

placed within the cavity with the plastic instrument. In Group C, the cavities

were filled with 0.5μL blood, then Endocem MTA was mixed and placed in

the cavity with a plastic instrument followed by GIC restoration. In Group D,

the cavities were filled with 0.5μL blood followed by Neo MTA, which was

mixed and placed within the cavity with a plastic instrument. After which the

access cavities were restored with type IX GIC. An immediate postoperative

shade was obtained with the Spectrophotometer. The samples were then stored

in tap water at room temperature. Each group’s shade was measured after one

day, 1 week, 2 weeks and 3 weeks. ∆E values were obtained from the samples

and analysed.

∆E Measurements:

The Spectrophotometer is a device used to measure the shade of the

tooth. The conducting end is placed on the middle third of the tooth’s labial

surface and the device is activated to measure the ∆E value. This value

consists of L (Lightness), c (chroma), h (hue) measurements.

L*c*h = ∆E

29

Statistical Analysis:

One -way ANOVA (analysis of variance) – to assess significant

differences between the tested materials.

Bonferroni multiple - comparison test

Figures

Figures

FIGURE 1: EXTRACTED HUMAN UPPER CENTRAL INCISORS

Figures

FIGURE 2: DECORONATION OF TEETH AT CEJ

FIGURE 3: DIAMOND DISC

Figures

FIGURE 4: HANDPIECE

FIGURE 5: ISO DIAMOND CYLINDRICAL BUR – SR -13 (MANI)

Figures

FIGURE 6: SYRINGE (UNILOK)

FIGURE 7: SALINE

Figures

FIGURE 8: SODIUM HYPOCHLORITE IRRIGATION SOLUTION

3% (CHEMDENT)

FIGURE 9: 17% EDTA SOLUTION (DESMEAR)

Figures

FIGURE 10: MICROPIPETTE

FIGURE 11: MTA (MICRO ANGELUS)

Figures

FIGURE 12: ENDOCEM MTA (MARUCHI)

FIGURE 13: NEO MTA PLUS (AVALON BIOMED)

Figures

FIGURE 14: GLASS IONOMER CEMENT TYPE IX (GC), MIXING

PAD AND AGATE SPATULA

FIGURE 15: GLASS SLAB AND CEMENT SPATULA

Figures

FIGURE 16: PLASTIC INSTRUMENT

FIGURE 17: CAVITY PREPARATION

Figures

FIGURE 18: SPECTROPHOTOMETER

Figures

FIGURE 19: MANIPULATION OF MTA ANGELUS

FIGURE 20: MANIPULATION OF ENDOCEM MTA

Figures

FIGURE 21: MANIPULATION OF NEO MTA

Figures

FIGURE 22: BLOOD ONLY

GROUP FIGURE 23: MTA ANGELUS

GROUP

FIGURE 24: ENDOCEM MTA

GROUP FIGURE 25: NEO MTA

GROUP

Figures

A

B

C

FIGURE 26: POSTOPERATIVE SAMPLES

D

Results

Results

30

RESULTS

The study was designed to evaluate the discolouration potential of

newer and improvised MTA materials when it comes in contact with blood

and to compare the discolouration potential among them. The discolouration

in teeth were measured with a spectrophotometer and the obtained ∆E values

were analysed with the IBM.SPSS statistics software 23.0 Version.

To find the significance difference one-way ANOVA was used and for

repeated measures, ANOVA with Bonferroni test was used for multiple

comparison.

The experimental groups were divided into four groups:

Group A – Blood only

Group B – Blood +MTA Angelus

Group C – Blood + Endocem MTA

Group D – Blood + Neo MTA

Table 1 represents the multiple comparison test analysis for the

materials used and measured at various intervals by one-way ANOVA. The

table shows the mean and SD of each group at measured intervals. The

immediate postoperative p value is 0.421(p>0.05), Group A(Blood only)

shows a mean value [21.39±1.34], Group B (MTA Angelus) shows a mean

value [20.20±4.37], Group C ( Endocem MTA) shows a mean value

Results

31

[21.57±0.77] and Group D (Neo MTA) shows a mean value [21.90±1.15].

After day 1 the p value is 0.445(p>0.05), Group A shows a mean value of

[21.35±1.38], Group B shows a mean value of [22.67±4.28], Group C shows a

mean value of [21.40±0.80] and Group D shows a mean value of

[21.50±1.09]. The p value after one week is 0.364(p>0.05), Group A shows a

mean value of [22.63±1.37], Group B shows a mean value of [22.59±3.79],

Group C shows a mean value of [21.86±0.74] and Group D shows a mean

value of [21.03±1.11]. After two weeks the p value is 0.264(p>0.05), Group A

shows a mean of [23.09±1.32], Group B shows a mean value of [22.79±3.89],

Group C shows a value of [22.16±0.75] and Group D shows a mean value of

[22.32±1.27]. After three weeks the p value is 0.316(>0.05), Group A shows a

mean of [23.88±1.26], Group B shows a mean value of [22.94±3.89], Group C

shows a mean value of [22.22±0.75] and Group D shows mean value of

[22.17±1.33].

Table 2 represents the repeated measures of ANOVA for Blood only

[Group A] (∆E). It shows a p value of 0.005 (P < 0.01) that is statistically

highly significant. The immediate postoperative measurement shows a mean

value of [21.39±1.34], day 1 shows a mean value of [21.88±1.38], after 1 week

shows a mean value of [22.63±1.37], after 2 weeks a mean value of

[23.09±1.32] and after 3 weeks it shows a mean value of [23.61±1.26].

Results

32

Table 3 shows pairwise comparison of blood only group at different

intervals. p value for all the group is 0.0005. The mean difference is

significant at the 0.05 level. The p value comparing immediate postoperative

with day 1 is 0.055, comparing with one week shows a value of 0.083,

comparing with two weeks shows a value of 0.104, comparing with three

weeks a value of 0.140. The p value comparing day 1 with one week is 0.073,

comparing with two weeks is 0.091, comparing with three weeks is 0.121. The

p value comparing one week with two weeks is 0.045 and comparing with

three weeks is 0.080. The p value comparing two weeks with three weeks is

0.049.

Table 4 shows repeated measures of ANOVA for MTA Angelus

[Group B] (ΔE). p value is 0.064 which is not statistically significant. The

statistical significance is at p>0.01. The immediate postoperative measurement

shows a mean value of [20.20 ±4.37], day 1 shows a mean value of

[20.53±4.28], after 1 week a mean value of [20.97±3.79], after 2 weeks mean

value of [21.12±3.89], after 3 weeks mean value of [21.25±3.89].

Table 5 shows pairwise comparison of MTA Angelus group at

different intervals. The p value comparing immediate postoperative with day 1

value is 0.053, compared after one week is 0.054, compared after two weeks is

0.014, compared after three weeks is 0.002. The p value comparing day 1

value with one week is 0.717, with two weeks is 0.137 and compared with

three weeks is 0.015. The p value comparing one week with two weeks is 1.00,

Results

33

comparing with three weeks is 0.016. The p value comparing two weeks with

three weeks is 0.675. The mean difference is significant at the 0.05 level.

Table 6 shows repeated measures of ANOVA for Endocem MTA

[Group C] (ΔE). p value is 0.0005 which is highly statistically significant at

the level of p<0.01. The immediate postoperative measurement shows a mean

value of [21.57 ±0.77], day 1 shows a mean value of [21.84±0.80], after 1

week shows a mean value of [22.02±0.74], after 2 weeks shows a mean value

of [22.16±0.75], after 3 weeks shows a mean value of [22.22±0.75].

Table 7 shows the pairwise comparison of Endocem MTA at different

intervals. The p value comparing the immediate postoperative value with day

one is 0.003, comparing with one week’s value is 0.0005, comparing with two

weeks is 0.0005, comparing with three weeks is 0.0005. The p value

comparing day 1 with one week is 0.077, comparing with two weeks is 0.006

and comparing with three weeks is 0.024. The p value comparing one week

with two weeks is 0.066, comparing with three weeks is 0.023. The p value

comparing two weeks with three weeks is 1.000. The mean difference is

significant at the 0.05 level.

Table 8 shows repeated measures of ANOVA for Neo MTA [Group

D] (ΔE). The p value is 0.003, that is highly statistically significant at p<0.01

level. The immediate postoperative measurement shows a mean value of

[21.90 ±1.15], after day 1 the mean value is [22.08±1.09], after one week the

Results

34

mean value is [22.18±1.11], after two weeks the mean value is [22.32±1.27]

and after 3 weeks the mean value is [22.36±1.33].

Table 9 shows the pairwise comparison of Neo MTA group at

different levels. The p value comparing immediate postoperative value with

day one shows value of 0.025, comparing with one week shows a value of

0.033, comparing with two weeks shows a value of 0.115 and comparing with

three weeks shows a value of 0.142. The p value comparing day 1 with one

week shows a value of 0.026, comparing with two weeks shows a value of

0.121 and comparing with three weeks shows a value of 0.149. The p value

comparing one week with two weeks shows a value of 0.130 and that

comparing with three weeks is 0.158. The p value comparing two weeks with

three weeks is 0.031. The mean difference is significant at the 0.05 level.

Graph 1, represents the ∆E mean values of immediate postoperative

measurement of all the groups.

Graph 2, represents the ∆E mean values after day 1 measurement of

all the groups.

Graph 3, represents the ∆E mean values after one week measurement

of all the groups.

Graph 4, represents the ∆E mean values after two weeks measurement

of all the groups.

Results

35

Graph 5, represents the ∆E mean values after three weeks

measurement of all the groups.

Graph 6, represents the ∆E mean values of the blood only group at

various intervals.

Graph 7, represents the ∆E mean values of MTA Angelus group at

various intervals.

Graph 8, represents the ∆E mean values of Endocem MTA group at

various intervals.

Graph 9, represents the ∆E mean values of Neo MTA group at various

intervals.

From table 1 and graphs 1-5, it is inferred that all the tested groups

showed discolouration.

From table 2, 3 and graph 6, the results showed that, though the

Blood Only group showed minimal discolouration initially, it exhibited

maximum discolouration potential at the end of three weeks.

Table 4, 5 and graph 7 showed that an immediate increase in

discolouration potential of MTA Angelus was seen after one day, that

gradually increased after a week and sustained the discolouration till the third

week.

Results

36

Table 6, 7 and graph 8 showed that Endocem MTA showed

minimum discolouration initially after placement. Its discolouration was very

minimal compared to all the other tested groups. The discolouration gradually

increased and remained the same in 2nd

and 3rd

week. Over all it showed

minimum discolouration potential when compared to other groups at the end

of 3rd

week.

Table 8, 9 and graph 9 showed that Neo MTA exhibited the least

discolouration potential when measured at different intervals. Compared to the

other tested groups, it showed minimum discolouration initially and at the end

of 3weeks.

Tables and Graphs

Tables and Graphs

Multiple comparison by Oneway ANOVA

N Mean S.D

F- value

P- value

Blood only 10 21.39 1.34

∆E

IMMEDIATE

POST OP

MTA Angelus

10 20.20 4.37

0.962

0.421 # Endocem MTA

10 21.57 0.77

Neo MTA 10 21.90 1.15

Blood only 10 21.35 1.38

MTA 10 22.67 4.28

∆E 1 DAY Angelus

0.912 0.445# Endocem

10 21.40 0.80 MTA

Neo MTA 10 21.50 1.09

Blood only 10 22.63 1.37

MTA 10 22.59 3.79

∆E AFTER 1 WEEK

Angelus 1.093 0.364#

Endocem 10 21.86 0.74

MTA

Neo MTA 10 21.03 1.11

Blood only 10 23.09 1.32

MTA 10 22.79 3.89

∆E AFTER 2

WEEKS

Angelus 1.381 0.264 #

Endocem 10 22.16 0.75

MTA

Neo MTA 10 22.32 1.27

Blood only 10 23.88 1.26

MTA 10 22.94 3.89

∆E AFTER 3

WEEKS

Angelus 1.97 0.136 #

Endocem 10 22.22 0.75

MTA

Neo MTA 10 22.17 1.33

No Statistical Significance at P < 0.05 level

TABLE 1: MULTIPLE COMPARISONS OF THE GROUPS AT

INTERVALS BY ONE-WAY ANOVA

Tables and Graphs

REPEATED MEASURES OF ANOVA FOR BLOOD ONLY (∆E)

Mean

S.D

F-value

P-value

Pre OP 21.16 1.35

210

0.0005 **

Immediate

Post OP

21.39

1.34

Day 1

21.88

1.38

After 1 week 22.63 1.37

After 2

weeks

23.09

1.32

After 3 weeks

23.61

1.26

** Highly Statistical Significance at P < 0.01 level

TABLE 2: REPEATED MEASURES OF ANOVA FOR BLOOD

ONLY (∆E)

Tables and Graphs

Pairwise Comparisons

(I) DeltaE

MD (I-

J)

Std.

Error

P-value

95% C.I

Lower

Bound

Upper

Bound

Pre OP

Immediate post OP

-.230*

.026

.0005

-.333

-.127

Day 1 -.720* .071 .0005 -1.001 -.439

After 1 Week -1.470* .101 .0005 -1.870 -1.070

After 2 Weeks -1.930* .123 .0005 -2.416 -1.444

After 3 Weeks -2.450* .159 .0005 -3.080 -1.820

Immediate

Post OP

Day 1 -.490* .055 .0005 -.706 -.274

After 1 Week -1.240* .083 .0005 -1.569 -.911

After 2 Weeks -1.700* .104 .0005 -2.113 -1.287

After 3 Weeks -2.220* .140 .0005 -2.772 -1.668

Day 1

After 1 Week -.750* .073 .0005 -1.040 -.460

After 2 Weeks -1.210* .091 .0005 -1.571 -.849

After 3 Weeks -1.730* .121 .0005 -2.209 -1.251

After 1

week

After 2 Weeks -.460* .045 .0005 -.639 -.281

After 3 Weeks -.980* .080 .0005 -1.296 -.664

After 2

weeks

After 3 Weeks

-.520*

.049

.0005

-.714

-.326

* The mean difference is significant at the 0.05 level.

TABLE 3: PAIRWISE COMPARISONS OF BLOOD ONLY

GROUP AT VARIOUS INTERVALS

Tables and Graphs

Repeated measures of ANOVA for MTA angelus (∆E)

Mean S.D F-value P-value

Pre OP 19.66 6.12

4.199

0.064 #

Immediate

Post OP 20.20 4.37

Day 1 20.53 4.28

After 1 week 20.97 3.79

After 2 weeks 21.12 3.89

After 3 weeks 21.25 3.89

** Highly Statistical Significance at P < 0.01 level

TABLE 4: REPEATED MEASURES OF ANOVA FOR MTA

ANGELUS (∆E)

Tables and Graphs

Pairwise Comparisons

(I) DeltaE

MD

(I- J)

Std.

Error

P-

value

95% C.I

Lower Bound

Upper Bound

Pre OP

Immediate post OP

-.540 .606 1.000 -2.936 1.856

Day 1 -.870 .628 1.000 -3.353 1.613

After 1 Week -1.310 .774 1.000 -4.371 1.751

After 2 Weeks

-1.460 .744 1.000 -4.400 1.480

After 3 Weeks

-1.590 .743 .914 -4.526 1.346

Immediate

Post OP

Day 1 -.330 .084 .053 -.664 .004

After 1 Week -.770 .197 .054 -1.550 .010

After 2

Weeks -.920*

.190 .014 -1.672 -.168

After 3 Weeks

-1.050* .167 .002 -1.712 -.388

Day 1

After 1 Week -.440 .192 .717 -1.200 .320

After 2 Weeks

-.590 .179 .137 -1.296 .116

After 3 Weeks

-.720* .151 .015 -1.318 -.122

After 1

week

After 2 Weeks

-.150 .079 1.000 -.463 .163

After 3 Weeks

-.280* .059 .016 -.514 -.046

After 2

weeks

After 3

Weeks -.130 .056 .675 -.351 .091

* The mean difference is significant at the 0.05 level.

TABLE 5: PAIRWISE COMPARISONS OF MTA ANGELUS

GROUP AT VARIOUS INTERVALS

Tables and Graphs

Repeated measures of ANOVA for Endocem MTA (∆E)

Mean S.D F-value P-value

Pre OP 21.48 0.74

66.395

0.0005 **

Immediate

Post OP

21.57

0.77

Day 1 21.84 0.80

After 1 week 22.02 0.74

After 2

weeks

22.16

0.75

After 3

weeks

22.22

0.75

** Highly Statistical Significance at P < 0.01 level

TABLE 6: REPEATED MEASURES OF ANOVA FOR ENDOCEM

MTA (∆E)

Tables and Graphs

Pairwise Comparisons

(I) DeltaE

MD

(I-J)

Std.

Error

P-

value

95% C.I

Lower

Bound

Upper

Bound

Pre OP

Immediate post OP -.090 .041 .814 -.251 .071

Day 1 -.360* .065 .006 -.618 -.102

After 1 Week -.540* .040 .0005 -.698 -.382

After 2 Weeks -.680* .059 .0005 -.914 -.446

After 3 Weeks -.740* .067 .0005 -1.005 -.475

Immediate

Post OP

Day 1 -.270* .045 .003 -.447 -.093

After 1 Week -.450* .022 .0005 -.538 -.362

After 2 Weeks -.590* .046 .0005 -.771 -.409

After 3 Weeks -.650* .060 .0005 -.888 -.412

Day 1

After 1 Week -.180 .049 .077 -.374 .014

After 2 Weeks -.320* .059 .006 -.554 -.086

After 3 Weeks -.380* .085 .024 -.718 -.042

After 1

week

After 2 Weeks -.140 .037 .066 -.287 .007

After 3 Weeks -.200* .045 .023 -.377 -.023

After 2

weeks

After 3 Weeks

-.060 .048 1.000 -.248 .128

* The mean difference is significant at the 0.05 level.

TABLE 7: PAIRWISE COMPARISONS OF ENDOCEM MTA AT

VARIOUS INTERVALS

Tables and Graphs

Repeated measures of ANOVA for Neo MTA (∆E)

Mean S.D F-value P-value

Pre OP 21.78 1.14

7.751

0.003 **

Immediate Post OP

21.90

1.15

Day 1 22.08 1.09

After 1

week 22.18 1.11

After 2

weeks

22.32

1.27

After 3

weeks

22.36

1.33

** Highly Statistical Significance at P < 0.01 level

TABLE 8: REPEATED MEASURES OF ANOVA FOR NEO MTA (∆E)

Tables and Graphs

Pairwise Comparisons

(I) DeltaE

MD

(I-J)

Std.

Error

P-

value

95% C.I

Lower

Bound

Upper

Bound

Pre OP

Immediate

Post OP -.120 .101

1.00 0

-.519 .279

Day 1 -.300 .114 .402 -.749 .149

After 1 Week

-.400 .115 .107 -.857 .057

After 2 Weeks

-.540 .154 .098 -

1.147 .067

After 3 Weeks

-.580 .172 .125 -

1.262 .102

Immediate

Post OP

Day 1 -

.180*

.025 .001 -.279 -.081

After 1 Week

- .280*

.033

.000 5

-.409 -.151

After 2 Weeks

-.420 .115 .081 -.876 .036

After 3 Weeks

-.460 .142 .150 -

1.020 .100

Day 1

After 1 Week

-.100 .026 .057 -.202 .002

After 2 Weeks

-.240 .121 1.00

0 -.720 .240

After 3 Weeks

-.280 .149 1.00

0 -.869 .309

After 1

week

After 2 Weeks

-.140 .130 1.00

0 -.655 .375

After 3 Weeks

-.180 .158 1.00

0 -.806 .446

After 2

weeks

After 3 Weeks

-.040 .031 1.00

0 -.161 .081

* The mean difference is significant at the 0.05 level.

TABLE 9: PAIRWISE COMPARISON NEO MTA GROUP AT

VARIOUS INTERVALS

Tables and Graphs

GRAPH 1: REPRESENTING ∆E IMMEDIATE POSTOPERATIVE

VALUES OF THE GROUPS

GRAPH 2: REPRESENTING ∆E DAY 1 VALUES OF THE GROUPS

Neo MTA MTA angelus Endocem MTA Blood only

0.00

4.00

8.00

12.00

16.00

20.00

24.00 ∆E Immediate Post OP

Neo MTA MTA angelus Endocem MTA Blood only

0.00

4.00

8.00

12.00

16.00

20.00

24.00

∆E 1 Day

MEA

N

MEA

N

Tables and Graphs

GRAPH 3: REPRESENTING ∆E 1 WEEK VALUES OF THE GROUPS

GRAPH 4: REPRESENTING ∆E 2 WEEKS VALUES OF THE GROUPS

Neo MTA Endocem MTA MTA angelus Blood only

0.00

4.00

8.00

12.00

16.00

20.00

24.00

∆E After 1 week

Neo MTA Endocem MTA MTA angelus Blood only

0.00

4.00

8.00

12.00

16.00

20.00

24.00

∆E After 2 weeks

MEA

N

MEA

N

Tables and Graphs

GRAPH 5: REPRESENTING ∆E 3 WEEKS VALUES OF THE GROUPS

Neo MTA Endocem MTA MTA angelus Blood only

0.00

4.00

8.00

12.00

16.00

20.00

24.00

∆E After 3 weeks

MEA

N

Tables and Graphs

∆E of Blood only

24.00 23.50 23.00 22.50 22.00 21.50 21.00 20.50 20.00 19.50

∆E of MTA angelus

21.50

21.00

20.50

20.00

19.50

19.00

18.50

GRAPH 6: REPRESENTING ∆E VALUES OF BLOOD ONLY GROUP

AT VARIOUS INTERVALS

GRAPH 7: REPRESENTING ∆E VALUES OF MTA ANGELUS

GROUP AT VARIOUS INTERVALS

Mea

n

Mea

n

Tables and Graphs

∆E of Endocem MTA

22.40

22.20

22.00

21.80

21.60

21.40

21.20

21.00

∆E of Neo MTA

22.50 22.40 22.30 22.20 22.10 22.00 21.90 21.80 21.70 21.60 21.50 21.40

GRAPH 8: REPRESENTING ∆E VALUES OF ENDOCEM MTA GROUP AT

VARIOUS INTERVALS

GRAPH 9: REPRESENTING ∆E VALUES OF NEO MTA GROUP AT

VARIOUS INTERVALS

Mea

n

Mea

n

Discussion

Discussion

37

DISCUSSION

The aim of this study was to compare the potential discolouration

effect of MTA Angelus, Endocem MTA and Neo MTA on natural teeth.

MTA is used in pulp capping, pulpotomy, regenerative endodontic

procedures and perforation repairs. One major drawback is the discoloration

potential when it interacts with blood, especially when placed in the anterior

teeth, which is of a discernible concern. Hence the study was carried out

constituting three newer and improvised MTA materials. Group A – Blood only

(control), Group B – Blood + MTA Angelus, Group C – Blood + Endocem

MTA and Group D – Blood + Neo MTA, wherein MTA is placed in direct

contact with blood.

Discolouration effect of Pro root MTA and MTA Angelus are

identified and confirmed by previous studies.48,17,3 The reason for these

discolourations have been widely discussed and proved by various authors. A

new version of MTA with modification in the composition have been recently

introduced3,49,11, which are Endocem MTA and Neo MTA. Endocem MTA has

been evaluated for its ability to discolour tooth by Ji-Hyun Jang et al 35. PubMed

search on Neo MTA resulted in 13 articles but Neo MTA has not been tested

for its ability to discolour teeth in clinical conditions,15 hence in this study we

are testing the discolouration potential of newer MTA(Neo MTA) along with

Endocem MTA and MTA Angelus.

Discussion

38

Discolouration in teeth are usually measured by visual assessment

with a shade guide and by computer analysis of digital images.50 However,

commercially available shade guides contain a limited selection of colours when

compared to those found in natural teeth 51 and visual shade matching is affected

by variable viewer interpretation and environmental influences.52 Recently

electronic devices such as colourimeters and spectrometers are being used for

measurement of tooth discolouration. Colourimeter is an instrument for

psychophysical analysis, which measures human eye-brain perception. A

spectrometer separates and measures spectral components of a physical

phenomenon. It measures a continuous variable of a phenomenon where the

spectral components are somehow mixed, such as a beam of white light.

Spectrometers are of two types: Spectrophotometer and Spectroradiometer. A

spectroradiometer is a light measurement tool that is able to measure both the

wavelength and amplitude of the light emitted from a light source. The

colour and translucency values of irregularly shaped objects are measured by

means of a spectroradiometer. Spectroradiometer provides clinically relevant

values because this measurement can eliminate the edge-loss effect, closely

simulate clinical viewing conditions, and also provide optical values of a

specified area.53 Spectrophotometer is an instrument for physical analysis,

which analyses the reflecting transmitting properties of objects without human

interpretation. According to Ishikawa-nagai et al, spectrophotometers,

generally, can provide more systematic and precise measurements than

colourimeters because of their ability to measure the amount of light reflected

Discussion

39

from objects throughout the visible spectra range.54 This is because a colour-

measuring instrument with computerized colour analysis allows standardized

and accurate colour matching compared with other conventional techniques.

In the present study a spectrophotometer was used for analysis of

the tooth discolouration. A spectrophotometer measures the spectral reflectance

or transmittance curve of an object. Light is released from a light source in the

spectrophotometer and then dispersed by a prism into a spectrum of different

wavelengths between 380-780 nm. The spectrophotometer then measures the

amount of the light reflected from the specimen for each wavelength in the

visible light and converts the data into numerical values of colour coordinates

CIE L*, c* and h*.55 It measures the space coordinates on any spectrum. The

main advantage of spectrophotometer over spectroradiometer is that they have

a stable light source (LED). Because of which the spectrophotometer was used

for this study.56 In the CIE L*c*h system, the idea was to create a linear colour

space in which the distance between the points defining individual colours

would be proportional to the perceptual difference between them (perceptual

colour spaces) and to present colours with the coordinates describing one of

their key attributes, ie: lightness, chroma (saturation) and hue (ie. L, C, h).57

ΔE is the colour stimulus that can be represented as a point in space, it is

calculated as the distance between the points representing these stimuli.

The change ΔE of each specimen was calculated using the following equation

Discussion

40

ΔE =[(ΔL)2+(ΔC)2+(ΔH)2]1/2

Where ΔL parameter indicates lightness and ranges in value from 0 (black) to

100 (white), ΔC indicates green/red. Here negative value indicates green and

positive value indicates red and ΔH indicates blue/yellow where blue value

indicates negative value and yellow indicates positive value.

In this study the Vita Easyshade Compact (Vita Zahnfabrik,

BadSackingen, Germany), which is a cordless, small, portable, cost efficient,

battery operated, contact-type spectrophotometer that provides enough shade

information to help aid in the colour analysis process. Different measurement

modes are possible with Easyshade Compact: tooth single mode, tooth area

mode (cervical, middle and incisal shades), restoration colour verification

(includes lightness, chroma and hue comparison) and shade tab mode

(practice/training mode).58

40 upper human central incisors were taken as the sample, with 10

teeth in each group. Anterior teeth were selected because they had wide root

canal diameter which would facilitate placement of the test materials easily1 and

discolouration is of more concern in the anterior teeth than posterior teeth. Also

MTA is frequently used in anterior teeth as they are commonly involved in

traumatic injury and may require vital pulp therapy procedures like pulp

capping, pulpotomy, regenerative procedures and apexification.59,60 Human

teeth were preferred for better reproducibility and clinical significance.21

Previous studies have reported the use of bovine teeth, because they have

Discussion

41

increased number of dentinal tubules next to the pulp, that simulate the human

teeth (21,343 ± 7290 per mm2 ± SD for human permanent teeth and 20,980 ±

4198 for bovine teeth).61,62,63,64 According to Schilke et al, bovine incisor crown

dentine is a suitable substitute for human teeth, whereas bovine incisor root

dentine appears less suitable due to its significantly higher tubule density.61

Whole blood was collected from a volunteer by venipuncture. The

collection tubes were sterile and spray coated with the anticoagulant K2EDTA

to prevent clotting in order to facilitate the experiment 13,4,65. Blood was placed

with a micropipette in the prepared cavities of all sample teeth, followed by their

respective group materials, Group A – Blood only, Group B – Blood followed

by MTA Angelus placement, Group C – Blood followed by Endocem MTA,

and Group D – Blood followed by Neo MTA placement. The pH of blood is

7.4. A study by Namazikhah reported that MTA when in contact with butyric

acid (byproduct of anaerobic bacteria metabolism, used to simulate the clinical

environmental conditions of periradicular infection) had distinctive crystalline

structures embedded within a more uniform matrix partially covered by

colloidal gel that may have been involved in the bonding of the various phases

of the cement.66 A low pH could potentially inhibit the setting reaction or

increase the solubility of calcium silicate-based materials, which could affect

the mechanical properties of the material including the surface microhardness.

Studies conducted by Elnaghy et al 67 and Namazikhah et al 66 stated that MTA

might be more sensitive to acidic pH environment inhibiting crystallization in

Discussion

42

hydration reaction.68 MTA has an initial pH around 10.2 that further increases

to 12.5 which is an alkaline setting environment.13 Surfaces exposed to a pH

value of 7.4 showed an amorphous poorly crystallized superficial gel structure

containing globular aggregate particles and microchannels. But these structures

disappeared when the cement hardened in an acidic environment with a

selective loss of matrix.67

After the placement of the respective materials, the access of the teeth was

restored with type IX glass ionomer cement and an immediate postoperative

measurement was taken. This was done to find out which group reported

discolouration immediately after the procedure. The teeth were then stored in

tap water (Tap water has a pH of 7.4 to 7.79 and an osmolality of 30 mOsm/kg)69

and the following measurements were taken after a day, 1 week, 2 weeks and 3

weeks. The ∆E values for all the groups at each interval were noted.

The collected data were analysed with IBM.SPSS statistics software 23.0

Version. To describe about the data descriptive statistics frequency analysis was

used, percentage analysis was used for categorical variables and the mean &

S.D were used for continuous variables. To find the significant difference

between the multivariate analysis the one-way ANOVA was used and for

repeated measures ANOVA was used with Bonferroni correction to control the

type I error on multiple comparison. The results of the present study showed

that there was statistically significant difference of the tooth in all coordinates

(L*, c*, h*) for all the 4 groups tested at all the time intervals. The amount of

Discussion

43

change in the value of color shift as measured by color difference (ΔE) was also

found to be statistically significant.

All the tested groups showed some amount of discoloration with

varying percentage at the measured intervals (Table 1). From Table 2, it is

observed that ΔE value measured preoperatively is 21.16(mean), postoperative

measurements - immediately is 21.39 (mean), after one day it is 21.88(mean),

after a week it is 22.63(mean), after two weeks a value of 23.09(mean) and after

3 weeks it shows a mean value of 23.61(mean). From the above values it is

inferred that the blood only group showed the maximum discolouration at the

end of 3 weeks. This is substantiated by a study conducted by Marin et al, where

he concluded that the major cause of discolouration was the accumulation of

hemoglobin or other forms of hematin molecules such as hemosiderin, hemin,

hematin and haematopinid.21,70,71,72 The flowing of blood components into the

dentinal tubules could be a possible reason for the maximum discolouration

potential in this group.1 The pigments responsible for discolouration are found

within the dentinal tubules and not in the intertubular dentin.21 According to

Marin et al, majority of the blood staining was confined to dentine but there was

also some discoloration of the enamel produced. This enamel staining was not

as pronounced as that observed in the coronal and radicular dentine; rather, it

appeared as a pinkish hue. It may be determined by the length of exposure of

the blood pigments to the tooth structure. The hemolysis and catabolism of

erythrocytes in the pulp chamber differ from other parts of the body as the pulp

Discussion

44

chamber is surrounded by dentine and cementum, which isolates it from any

inflammatory or healing response from adjacent tissues.21

When blood comes in contact with MTA the discolouration

potential is exaggerated, though, not as pronounced as in the blood only group.4

From Table 4, the preoperative ΔE value 19.66(mean) shows a significant

difference immediately after the placement of MTA Angelus of ΔE value

20.20(mean), that sustains the increase in discolouration even after day 1 (ΔE-

20.53). After a week’s time the measured value is 20.97 and the following

week’s mean values are 21.12 and 21.25 respectively, that resulted in a

significant increase in the discoloration potential of MTA Angelus. Average

difference of 1.6 is observed from the preoperative ΔE value and postoperative

3rd week ΔE value. This signifies that when the material comes in contact with

blood, the discoloration potential is increased to a certain extent. The following

could be the reasons for discolouration: The presence of bismuth oxide in the

MTA Angelus formulation causes the discoloration.16,73 Collagen, present in

dentin matrix, reacts with bismuth oxide, resulting in a grayish

discoloration.17,37 Release of heavy metal ions from MTA and the slow setting

reaction of MTA permits the absorption and hemolysis of erythrocytes, resulting

in material discoloration and subsequent tooth discoloration when in contact

with blood. 4,74,75 MTA Angelus contains bismuth oxide as a radiopacifier which

could be a major contributing factor for its discolouration potential and its slow

Discussion

45

setting time provides contamination with blood, that makes the material porous

leading to less dimensional stability too.76,77

From table 6, the discoloration potential of Endocem MTA is

observed as follows, preoperative ΔE mean value is 21.48, immediate

postoperative mean value is 21.57, after one day the mean value is 21.84, one

week later the value is 22.02, after two weeks the value is 22.16 and after three

weeks the value is 22.22. A difference of 0.7% is observed from the immediate

postoperative measurement to the final week measurement, which shows the

discoloration potential of Endocem MTA to be least, followed by MTA Angelus

and blood only groups. Endocem MTA is composed of the tricalcium silicates,

and bismuth oxide, which is used as the radiopacifier.78 The unique property of

Endocem MTA is its rapid setting time and ease in manipulation.35 Furthermore,

the biocompatibility and osteogenicity of Endocem are similar to those of

conventional MTA.78 The small particle size of Endocem increases the surface

contact of the particles while mixing with sterilized water, resulting in faster

setting time and ease of manipulation.78,2 As the contact surface area increases,

the reactivity of the calcium silicate particles to form calcium hydroxide and

calcium silicate hydrate phases also increases.35 A study by Choi et al compared

the setting time of Endocem MTA with ProRoot MTA, which reported a final

setting time of 4 minutes ± 30 seconds for Endocem MTA and that of 261 ± 21

minutes for ProRoot MTA.78 The setting time of Endocem MTA provides a

Discussion

46

sufficient working time when performing retro- grade filling or perforation

repairs.11

From table 8, Neo MTA group showed the following ΔE mean

values, preoperative mean value is 21.78, immediate postoperative value is

21.90, after day one 22.08 is the value, one week later 22.18 is the value, after

the second and third week 22.32 and 22.36 are the values respectively. A

minimal difference of 0.5 is observed from the immediate postoperative

measurement and final week ΔE mean values, that signified the least

discolouration potential of Neo MTA when compared to MTA Angelus and

Endocem MTA. Studies have concluded that bismuth oxide as a radiopacifier

was a major factor for the discoloration potential of MTA, hence in Neo MTA

tantalum oxide was replaced as the radiopacifier.49 A study conducted by

Camilleri et al showed that Neo MTA had clusters rich in tantalum and oxygen

in the scanning electron micrograph images,15 this relatively produced lesser

discolouration and did not interfere in the hydration mechanism. Tantalum

oxide provides adequate radiopacity and showed least discoloration.15,79 Oh et

al also reported that tantalum is a biomaterial with applications in tissue

regeneration and has excellent radiopacity with low cytotoxicity

properties.80,79Aluminum has a strong effect on setting reaction of MTA. It

rapidly reacts with the formed calcium hydroxide in the presence of water

forming calcium aluminate hydrate. The presence of relatively high amount of

gypsum and aluminium in Neo MTA Plus accounts for its rapid setting as

Discussion

47

observed during mixing where it sets into a hard mass within few minutes,

whereas the setting of MTA Angelus takes several hours.49 Due to the faster

setting time of Neo MTA, the contamination of tissue fluids does not alter the

property of the set material. Even though when Neo MTA is placed in a low pH

environment, the faster setting time helps the material to attain the alkaline pH

which withstands washout and disintegration of the material.81,82

A comparative analysis of intergroups Group A and Group B (Blood

and MTA Angelus) revealed that on day 1, there was an immediate increase in

the ∆E value of MTA Angelus group when compared to the Blood only group.

This was in accordance with the study conducted by Felman and Parashos, that

contamination of MTA with blood exaggerated the discolouration potential

immediately. According to Marciano et al, bismuth oxide present in the

composition of MTA Angelus was a major discolouration contributing factor ,

furthermore, when contacted with sodium hypochlorite , it produced a dark

brown precipitate.83,5 Bismuth oxide undergoes oxidation and destabilises the

oxygen, which reacts with carbondioxide producing bismuth carbonate

resulting in a black precipitate.43,3,16

Blood when in contact with Group C material (Endocem MTA),

produced significantly lesser discoloration when compared to Group A (Blood

only). This is because of the hydration mechanism that differs due to its fine

particle size.35,78,2 Particle size ranges from less than 1μm to more than 30μm

and particle size of bismuth oxide in MTA was reported to be between 10 and

Discussion

48

30μm.84 Hydration of Endocem MTA results in decreased amount of free

calcium hydroxide and increase in the formation of stable crystals of calcium

silicate hydrate and calcium aluminate hydrate, which confers mechanical

strength of the material.14,25 Endocem MTA possessed increased washout

resistance due to its fast setting property without any chemical accelerators.78

Comparison of Group A (Blood only) with Group D (Neo MTA),

showed that Neo MTA results in the least discoloration with 1.87 standard

deviation. According to Felman, Neo MTA and Endocem MTA did not

discolour as severely as the blood only group as the material limited the

diffusion of erythrocytes from the canal into the coronal dentin by an increased

setting time.4,15 It has fine particles, which improves its handling characteristics

and increases the speed of hydration process.82 The presence of tantalum

pentoxide provided the adequate radiopacity and was inert as it did not produce

discoloration.15,85 Due to its high atomic number (72), inherently it possesses

the radiopaque property.86 Tantalum has a long history as a biomaterial and

used in dentistry.22,86 In a study where MTA plus and Neo MTA were compared

for alkaline activity, Siboni reported that the alkalinity of Neo MTA did not

decrease for about three days and only MTA plus showed a rapid decrease in its

alkalinity. This provides an antibacterial environment and also promotes apatite

nucleation.87

Comparison of Group B (MTA Angelus) with Group C (Endocem

MTA) and Group D (Neo MTA) showed that MTA Angelus showed the

Discussion

49

maximum discoloration, followed by Endocem MTA and Neo MTA. The

hydration mechanism of Endocem MTA and Neo MTA is different from that of

MTA Angelus. MTA Angelus has a similar hydration mechanism to that of

Portland cement. The setting of MTA usually occurs in 4 stages: Pre induction

period, Induction (dormant period), Acceleration period and Post acceleration

period.76,46,23 These stages were reduced to a 2stage mechanism, resulting in a

faster setting time from 3-4 hours to 10-15 mins.78,88 Neo MTA consists of

tricalcium and dicalcium silicates, calcium sulphate and tantalum oxide wherein

MTA Angelus consists of tricalcium silicate, dicalcium silicate, tricalcium

aluminate, calcium aluminoferrite and bismuth oxide.89,47,2,76 During the

hydration process Neo MTA does not produce ferrous oxide as the

aluminoferrite phase is not present and bismuth oxide is replaced by tantalum

oxide.47,15 Moreover minor components that caused discolouration on leaching

such as arsenic, lead and chromium are in lesser percentages in Neo MTA when

compared to MTA Angelus.33,90,91

On a comparative analysis of the inter groups, from table 1 it is

observed that all the tested groups showed a certain amount of discolouration in

a span of one month. Group A (Blood only) showed the maximum

discolouration, which is similar to the previous studies by Yoldas et al and

Marin et al.1,21 Marin et al observed the ability of blood components to

penetrate dentine and induce discolouration of enamel which are more

pronounced with longer exposure times. Although enamel has no tubular

Discussion

50

morphology, its organic structural features at the dentino-enamel junction, may

play a role in the discolouration process.21 According to Ahmed et al, the

erythrocytes in the pulp chamber or dentinal tubules degrade into haem

molecules and these release iron during hemolysis.70 Hattab et al suggested

that the iron released can be converted to black ferric sulphide with hydrogen

sulphide produced by bacteria and this may cause grey discolouration of the

tooth.71 Attin et al also suggested that the degrading proteins of necrotic pulp

tissue may also cause staining of the tooth.72 Group B (MTA Angelus) showed

an escalated discolouration at day 1 (Graph 3) and its discolouration potential

was sustained up to 3 weeks of the study. This is similar to other in-vitro and

in-vivo studies. According to Felman and Parashos, the slow hydrating

process of MTA Angelus may permit the absorption and subsequent hemolysis

of erythrocytes from the adjacent pulpal tissue, thus resulting in both material

and subsequent tooth discoloration.4 According to Yoldas et al and Marciano

et al, the possible mechanism of MTA Angelus discolouration may be due to

the oxidation and incorporation of the iron content within the MTA powder into

the calcium aluminoferrite phase of the set MTA cement.1 Additionally, the

release of heavy metal ions and the presence of bismuth oxide as a radiopacifier

within MTA could lead to discoloration.83 Valles et al reported in studies that

when bismuth oxide is exposed to high temperatures or light irradiation in an

oxygen-free environment, it undergoes dissociation to produce metallic bismuth

and oxygen. The reduced bismuth atoms form black crystals that darken the

tooth.16,73 Studies conducted by Camilleri et al reported that zirconium oxide

Discussion

51

as a radiopacifier in biodentine proved to be inert when compared to bismuth

oxide present in MTA Angelus.77 He also concluded in another study that

bismuth oxide affected the hydration mechanism, as it formed a part of calcium

silicate hydrate and also affected the precipitation of calcium hydroxide.76

Group C (Endocem MTA) showed discolouration after day one that did not

differ even after 3 weeks of the study.35,43 Choi et al stated that, longer setting

time led to the material washout due to its contact with tissue fluids before the

material could set, hence faster setting cement Endocem MTA may overcome

this drawback.78 In a study by Jang et al reported that Endocem MTA showed

an initial significant discolouration after one week but showed minimum

discolouration during the course of the remaining 11 weeks.35 Kim et al

reported that Endocem MTA up regulated OPN production that helped in wound

healing and inflammatory reactions when compared to Proroot MTA.11 Group

D (Neo MTA) showed the least discoloration at the end of three weeks.92,4,57 A

study conducted by Camilleri et al showed that Neo MTA had clusters rich in

tantalum and oxygen in the scanning electron micrograph images.15 According

to Shokouhinejad. Neo MTA has fine powder particles, which improves its

handling properties and increases the speed of hydration.82,23 In a study by

Walsh et al, Neo MTA Plus had similar effects on inflammation, pulp response,

periodontal ligament and cementum formation, and apical tissue healing in

dogs, also Neo MTA Plus had superior dentin bridge quality.81 Group C

(Endocem MTA) showed a little higher discoloration than Group D (Neo MTA)

throughout the study measurements (Graphs 2-6). On a descending order of

Discussion

52

discoloration produced by the groups from immediate postoperative to the third

week of the study is as follows, Group A (Blood only), Group B (MTA

Angelus), Group C (Endocem MTA) and Group D (Neo MTA). The percentage

of increase in the discoloration within each group is as follows, Group A (Blood

only) – 2.45%, Group B (MTA Angelus) – 1.59%, Group C (Endocem MTA) –

0.74% and Group D (Neo MTA) – 0.58%. (Graph 7-10)

Neo MTA is a novel calcium silicate cement with fine powder particles,

which improves its handling properties and increases the speed of hydration.82

When the material is placed in an acidic environment, the faster setting time

produces a set hard mass within minutes that provides increased washout

resistance. Hence in clinical situations such as root end fillings and perforation

repairs, this setting time is ideal. More over bismuth oxide replaced by tantalum

oxide, provides adequate radiopacity and does not interfere in the hydration

mechanism. Neo MTA produced desirable healing during inflammation and

reparative bone formation. It even displayed cementum and PDL reformation.81

This is the first study wherein we evaluated the discolouration potential of

this new novel material, Neo MTA in natural teeth. This study evaluated the

discolouration effect of the newer cement. This material exhibited

discolouration like other currently available material. After placement of the

material in contact with blood, it showed discolouration but very minimal when

compared to the currently available material. From this study it can be

Discussion

53

concluded that, it can be recommended as an ideal material for vital pulp

therapies and root end filling procedures.

Summary

Summary

54

SUMMARY

The purpose of this study was to compare and evaluate the

discoloration potential of three MTA materials, MTA Angelus, Endocem

MTA and Neo MTA in coronal portion of natural teeth when placed in contact

with blood using a Spectrophotometer.

The sample size collected was 40 human extracted upper central

incisors. The teeth were cleaned with pumice after which the crowns were

separated from their roots with a diamond disc and the crowns were retained

for the study. A cylindrical bur was used to prepare a cavity on the lingual

surface of the teeth of 3mm depth. The teeth were then rinsed and placed in

3% sodium hypochlorite solution for 30 minutes followed by 17% EDTA

solution for 2 minutes for smear layer removal. The teeth were then rinsed and

randomly divided into 4 groups: Group A - Blood only; Group B – Blood +

MTA Angelus; Group C – Blood + Endocem MTA; Group D – Blood + Neo

MTA. An initial shade was obtained using a spectrophotometer for all the

teeth. Each tooth was filled with blood using a micropipette, followed by the

testing material and their access cavity were restored with Type IX GIC. In

Group A, the cavities were filled only with 2.0μL blood followed by GIC

restoration. In Group B, the cavities were filled with 0.5μL blood followed by

MTA Angelus using a plastic instrument and restored with GIC. In Group C,

the cavities were filled with 0.5μL blood, then Endocem MTA was placed in

Summary

55

the cavity followed by GIC restoration. In Group D, the cavities were filled

with 0.5μL blood followed by Neo MTA and restored with GIC. An

immediate postoperative shade was obtained with the Spectrophotometer. The

samples were then stored in tap water at room temperature. Each group’s

shade was measured after one day, 1 week, 2 weeks and 3 weeks. ∆E values

were obtained from the samples and analysed.

The Spectrophotometer is a device used to measure the shade of the

tooth. The conducting end is placed on the middle third of the tooth’s labial

surface and the device is activated to measure the ∆E value. This value

consists of L (Lightness), c (chroma), h (hue) measurements.

L*c*h = ∆E

The collected data was analysed statistically by one-way ANOVA and

Bonferroni tests. The results showed P value = 0.0005 [P < 0.01] for the

Group A (Blood only) which is highly significant. P value = 0.064 [P > 0.01]

for Group B (MTA Angelus), P value = 0.0005 [P < 0.01] for Group C

(Endocem MTA) which has high statistical significance and P value = 0.003

[P < 0.01] for Group D (Neo MTA). All the tested group showed

discolouration. The blood only group showed maximum discolouration

initially and at the end of 1st week, 2

nd week, 3

rd week. The MTA Angelus

group showed maximum discolouration (22.9±3.89). Initially after placement,

increase in discolouration is observed after day 1 (22.6±4.28), after one week

the value is (22.59±3.79), after two weeks (22.79±3.89) and third week

Summary

56

(22.94±3.89) which showed its discolouration potential. MTA Angelus

showed maximum discolouration at the end of 3rd

week. Endocem MTA

showed discolouration but less when compared to the blood and MTA

Angelus group. The postoperative value after the placement is (21.40±0.80), at

the end of 1st week (22.02±0.74), 2

nd week (22.16±0.75), 3

rd week

(22.22±0.75). There is an initial discolouration which is less when compared

to Group A and Group B. Neo MTA showed very minimal discolouration. The

initial discolouration after placement is (21.90±1.15) which is the least when

compared to Group A, Group B and Group C. The 2nd

and 3rd

week values

(22.32±1.27) and (22.36±1.33) respectively also showed least discolouration

when compared to the other groups. On a concluding note, among the four

groups, blood only group (Group A) showed the maximum discoloration at all

the stages and reached its maximum after the 3rd

week. When compared to

other groups, MTA Angelus (Group B) showed marked discolouration, though

it was not higher than that of blood only (Group A). After one day it showed

significant discolouration (higher than the blood only group) and continued to

increase its discolouration. Discolouration was observed after 1st and 2

nd week

and was higher after the 3rd

week. Endocem MTA showed minimal

discoloration. After one day Endocem MTA exhibited less discolouration

when compared to other groups. The minimal discolouration remained the

same in the 1st, 2

nd and 3

rd week (did not exhibit sustained discolouration

potential). Endocem MTA when compared to Neo MTA exhibited slightly

higher discolouration potential. Neo MTA (Group D) showed the least

Summary

57

discolouration when compared to other groups. Initially after day one Neo

MTA showed less discolouration. After the 1st week the discolouration was

sustained without any increase. This trend continued in the 2nd

and 3rd

week.

The discolouration potential is as follows in the descending order, blood only

(Group A) > MTA Angelus (Group B) > Endocem MTA (Group C) > Neo

MTA (Group D). The newer MTA, Neo MTA can thus be used for vital pulp

therapies and regenerative procedures especially in anterior teeth where

esthetic is of a concern with ease of handling and better outcomes.

Conclusion

Conclusion

58

CONCLUSION

1. All the four groups, Blood only, Blood + MTA Angelus, Blood +

Endocem MTA and Blood + Neo MTA used in this study showed

discolouration.

2. Blood only group showed discolouration immediately. The

discolouration gradually increased after day one and progressed till the

end of first week. The same trend continued till 2nd

week and reached

its maximum in the 3rd

week.

3. MTA Angelus group showed a significant increase in discolouration

immediately. especially after day one. After one week its

discolouration potential gradually increased. Discolouration increased

significantly in the 2nd

week. It showed a marked discolouration

potential after the 3rd

week of the study.

4. Endocem MTA group showed minimum discolouration after initial

placement and a very minimal increase in discolouration was observed

after day one. The discolouration potential after one week was

minimum. The discolouration produced is sustained in the 2nd

week. Its

discoloration potential after the 3rd

week did not show any significant

increase.

5. Neo MTA showed very minimal discolouration initially. Further

increase in its discolouration potential was not observed after day one.

Conclusion

59

The attained discolouration remained the same in the 1st

week.

There was no significant increase in discolouration after 2nd

week.

Discolouration potential of Neo MTA was very minimum after the 3rd

week.

6. Blood only group when compared to MTA Angelus, showed lesser

discolouration initially. But at the end of 3 weeks Blood only group

exhibited maximum discolouration. MTA Angelus showed an

immediate increase in discolouration after day one that was higher than

that of blood only group. But towards the 3rd

week, MTA Angelus

exhibited lesser discolouration than blood only group.

7. Blood only group when compared to Endocem MTA and Neo MTA

groups exhibited the maximum discolouration initially, after one day,

one week, two weeks and three weeks.

8. MTA Angelus compared to Endocem MTA showed maximum

discolouration when the material was placed in contact with blood.

Throughout the study Endocem MTA exhibited minimal

discolouration when compared to MTA Angelus.

9. MTA Angelus when compared to Neo MTA group showed maximum

discolouration initially and up to the 3rd

week of study.

10. Endocem MTA when compared to Neo MTA exhibited slightly

increased discolouration. Initially both the groups showed more or less

the same discolouration potential. By the end of one week Neo MTA

Conclusion

60

exhibited least discolouration when compared to Endocem MTA and

remained the same till the 3rd

week.

11. On an ascending order of discolouration potential of the groups tested

is as follows,

Neo MTA > Endocem MTA > MTA Angelus > Blood only group

12. Spectrophotometer served as a source for discoloration analysis which

showed reliability.

This is the first study wherein we evaluated the discolouration potential

of this new novel material, Neo MTA in natural teeth. This study evaluated the

discolouration effect of the newer cement. This material exhibited

discolouration potential like any other currently available MTA group of

materials. After placement of the material in contact with blood, it showed

discolouration but very minimal when compared to the currently available

material. From this study it can be concluded that, it can be recommended as

an ideal material for vital pulp therapies, root end filling procedures,

perforation repairs and where ever the material is in contact with blood.

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Annexures

Annexures

ANNEXURE – I

Annexures

ANNEXURE –II