COMPARISON OF INTRACANAL CALCIUM HYDROXIDE, MINERAL ...

COMPARISON OF INTRACANAL CALCIUM HYDROXIDE, MINERAL

TRIOXIDE AGGREGATE AND PORTLAND CEMENT TO INDUCE pH CHANGES

IN SIMULATED ROOT RESORPTION DEFECTS IN HUMAN TEETH- AN

INVITRO STUDY

Dissertation Submitted to the

Rajiv Gandhi University of Health Sciences, Bengaluru, Karnataka

In partial fulfillment

of the requirements for the degree of

MASTER OF DENTAL SURGERY

In

CONSERVATIVE DENTISTRY

&

ENDODONTICS

By

Dr. SARITA BHANDARI

Under the guidance of

Dr. MASHALKAR SHAILENDRA MDS

Professor & HOD

DEPARTMENT OF

CONSERVATIVE DENTISTRY & ENDODONTICS

AL-BADAR RURAL DENTAL COLLEGE AND HOSPITAL,

GULBARGA

2017-2020

LIST OF ABBREVIATIONS

SYMBOLS ABBREVIATIONS

ANOVA

Analysis of variance

ATP-ase

Adenosine triphosphate

Ca(OH)2

Calcium hydroxide

CEJ

Cemento Enamel Junction

EDTA

Ethylene diamine tetra acetic acid

hr

Hour

K file

Kerr file

min

Minute

MMP

Matrix MetalloProteinease

MTA

Mineral Trioxide Aggregate

NaOCl

Sodium hypochlorite

PDL

Periodontal ligament

TRAP enzymes

Tartrate resistant acid phosphate enzymes

wk

Week

ZnOE

Zinc oxide eugenol

LIST OF TABLES

TABLE

NO

LEGENDS

PAGE NO

1

Summary of pH values of each experimental and control

group

31

LIST OF FIGURES

FIGURE NO

LEGENDS

PAGE NO

1

Teeth specimen

57

2

Radiographs for confirmation of single canal (a)

parallel angulation (b) mesial angulation (c) distal

angulation

58

3

Decoronated specimen

59

4

Shaping of canals

60

5

Cavity preparation at 5 mm coronal to the apical

foramen

61

6

Radiograph for confirmation of voidless placement

of materials

61

7

Prepared samples in scintillation vial

62

8

Digital pH meter

63

ABSTRACT

ABSTRACT

AIM:

Comparison of intracanal Calcium Hydroxide, Mineral Trioxide Aggregate and Portland

Cement to induce pH changes in simulated root resorption defects in human teeth -an in vitro

study.

MATERIALS AND METHOD:

100 extracted teeth were decoronated with a standard length of 14 mm. Root canal

preparation was performed by using Pro Taper rotary system. To simulate root resorption

defect on the buccal surface of root, a cavity preparation was done at 5 mm from apex (1.2

mm diameter and 0.6 mm deep). Teeth were randomly equally divided and filled with

Calcium hydroxide (n=30), MTA (n= 30), Portland cement(n=30), Control group(n=10 teeth).

Successful placement was evaluated with radiographs. Subsequently, pH measured after

every 20 min, 3 hours, 24 hours, 1 week, 2 weeks, 3 weeks, 4 weeks of each tooth with

calibrated digital pH meter.

STATISTICAL ANALYSIS:

ANOVA with Tukey test was performed to analyse the difference in pH of three intracanal

filling material in different intervals of time for four weeks.

RESULTS:

All the experimental groups namely Ca(OH)2 , MTA and Portland cement in root surface

cavities showed significant pH changes during 4 week experimental period individually.

MTA and Portland cement showed higher alkaline pH changes in comparison to Ca(OH)2.

ABSTRACT

The Portland cement showed highest alkaline pH changes among all the groups at the end of

4 week experimental period.

CONCLUSION:

The pH changes in root surface cavities of Portland cement were highest in comparison with

MTA and calcium hydroxide during the 4 week experimental period. These results indicate

that it may be efficacious to use Portland cement in root resorption cases.

KEY WORDS: Analysis of variance, Calcium hydroxide, Mineral trioxide aggregate,

Portland cement, Root resorption

INTRODUCTION

1

INTRODUCTION

Root resorption can be characterized as one of the reason for progressive destruction of dental

hard tissue. Root resorption is process which can be described as the condition associated

with either a physiological or pathological process ending in a loss of tissue such as dentin,

cementum and alveolar bone. Physiological root resorption which is associated with primary

teeth is desirable because it results in exfoliation of the deciduos teeth, thereby allowing

eruption of the permanent teeth. However, root resorption of permanent dentition is

unfavourable because it might result in irreversible damage and or eventual tooth loss.

Root resorption might be classified by its location i.e., internal or external resorption. Internal

root resorption is initiated within pulp tissue whereas external root resorption starts in the

periodontal ligament and is classified by location as apical, lateral or cervical.

Internal root resorption has an uncommon occurrence and its etiology is also poorly

understood. The predisposing factors for internal root resorption can be trauma, pulpitis,

pulpotomy, cracked tooth, tooth transplantation, restorative procedures, orthodontic treatment

or Herpes zoster viral infection.1

The etiology behind the external root resorption of permanent teeth is usually the result of

trauma, chronic inflammation of pulp/periodontal tissues or both, induced pressure in

periodontal ligament (orthodontic movement), tumours or tooth eruption.2

Pulpal infection is the commonest stimulation factor for root resorption. The injuries to

precementum or predentin or infected dentinal tubules stimulate the inflammatory process

within osteoclasts in the periradicular tissues or in pulpal tissues leading to external or

internal resorption.

INTRODUCTION

2

Periodontal infection root resorption can cause injury of precementum which is apical to

epithelial attachment and followed by bacterial stimulation that originates from the sulcus can

cause the external root resorption.

Orthodontic pressure root resorption can lead to continuous pressure on the root results in

injury which stimulates the resorbing cells in the apical third of the roots leading to

resorption.

Impacted tooth or tumour pressure during eruption of permanent dentition or tumour

impinging on the root of the tooth and may cause pressure root resorption.

Ankylotic root resorption can occur in intrusive luxation or avulsion (extended dry time

outside mouth) injuries, the healing may occur with bone surface without any intermediate

attachment called “dento alveolar ankylosis”. Osteoclasts are directly in contact with

mineralized dentin in the exposed root surface. Hence, bone is laid down instead of dentin.

External Tooth resorption takes place by four ways:

1. Destruction of cementoblasts from external surface of a root by leaving periodontal

structures alive, with varying inflammatory degrees

2. Exposure of dentine gaps in CEJ, leaving alive the other gingival structures that cover it,

within inflammation degrees.

3. Destruction of odontoblasts in external surface of root, leaving other pulpal structures

alive, within inflammation degrees.

4. Simultaneous destruction of epithelial remains of malassez and cementoblasts in external

surface of the root, with necrosis or elimination of periodontal ligament.3

INTRODUCTION

3

The cellular pathway for root resorption starts by

1) Clastic cell adhesion in external root surface

2) Clastic cell fusion and activation of molecular pathway

3) Regulatory mechanism.

Periodontal ligament and bone marrow derived circulating mononuclear hematopoietic

precursor cells directs odontoclastic differentiation (Hienz et al). Cell commitment and

mononuclear cell fusion are regulated by E- cadherin which is required for cell to cell

adhesion. Activated osteoclastic cells get attached to mineral matrix, forming a sealing zone

and adopting a polarized morphology which contains ruffled border and secrete proteases

which initiate mineral resorption. V-ATPase pump carries the protons produced by carbonic

anhydrase II to these ruffled border membrane and releases them into the resorption pit and

generate an acidic microenvironment which is completed by chloride transport. TRAP

enzymes are responsible for endocytosed material elimination. Finally, end results of

resorption process is degradation of the organic component.4 The MMPs and cathepsins are

responsible for degradation of collagen rich organic bone matrix (Teitbaum 2007).

The optimal condition for resorption to take place is an acidic pH. A well known feature of

the odontoclasts and osteoclasts is the proton pump, pumping hydrogen ions sealed

compartment and thus intensifying acidic environment.5,6 Moreover, at an acidic pH, the acid

hydrolases are active, and the demineralization occurs. DeDuve and Wattiaux reported that

the alkaline pH would be unfavorable for osteoclastic acid hydrolase activity. An alkaline pH

prevents dissolution of mineral component and might also activate alkaline phosphatases,

which is important for hard tissue formation.7,8

INTRODUCTION

4

Root resorption can be arrested by proper endodontic therapy.9 Traditionally calcium

hydroxide has been used as an intracanal medicament for treatment of external inflammatory

root resorption.8 The high alkaline pH of calcium hydroxide has the ability to not only kill

micro-organisms but also to inhibit osteoclastic activity thus preventing dissolution of the

mineral components and creating a favourable environment for hard tissue formation.10

Mineral trioxide aggregate (MTA) has been used for vital pulp therapy, root end filling,

apexification and perforation repairs. It is biocompatible and can perfectly seal dentin.

Torabinejad et al reported that MTA activates cementoblasts matrix formation due to its high

pH, or by releasing substances that activate cementoblasts. In comparision with calcium

hydroxide it shares similar property of high alkaline pH and inhibition of microorganisms.

Further calcium hydroxide has disadvantage of increased risk to fracture. Hence MTA

provides as very good alternative.11

MTA’s chief ingredient is Portland cement.12 Funteas et al analysed the samples of MTA and

Portland cement for different elements and revealed that there was significant similarity

among them except there was absence of bismuth in Portland cement.13 In 2003, Saidon et al

analysed the in vitro cytotoxicity and tissue reactions of MTA and Portland cement but found

no difference in cellular reactions.14 Abdullah et al investigated the biocompatibility of MTA

and Portland cement and showed that Portland cement is biocompatible and may have a bone

healing promoting factor.15 Therefore, due to the low cost of Portland cement and similar

properties as compared to MTA, Portland cement can be considered as a possible substitute.

Various studies have been done on diffusion of hydroxyl ions from several intracanal

medicaments and MTA have shown significant results with high amount of alkaline pH but

MTA is expensive. In order to search for other cost effective alternatives which also possess

INTRODUCTION

5

similar properties, it is necessary to assess the comparative properties of newer materials.

Portland cement is not only the chief constituent of MTA but also can be cost effective.

A variety of methods have been used to measure the hydroxyl ions diffusion through dentine.

Tronstad et al 1980 used pH indicating papers or solutions but they have limited accuracy and

difficult to interpret. Wang and Hume 1988 used pH measurement of ground dentine and

Fuss et al 1989 used pH measurement of surrounding medium but it had limited accuracy.

Larsen and Horsted-Bindslev 2000 used high impedence pH meter with a pH measuring

electrode and found it the most accurate approach with numerical records.

Thus this study “Comparison of intracanal Calcium hydroxide, Mineral trioxide aggregate

and Portland cement to induce pH changes in simulated root resorption defects in human

teeth -An in vitro study” was proposed to compare the pH changes in Calcium hydroxide,

MTA and Portland cement.

AIM AND OBJECTIVES

6

AIM AND OBJECTIVES

Aim-

Comparison of intracanal Calcium Hydroxide, Mineral Trioxide Aggregate and Portland

Cement to induce pH changes in simulated root resorption defects in human teeth

Objectives-

1. To evaluate pH changes in simulated root resorption defect in Calcium Hydroxide group

during 4 weeks.

2. To evaluate pH changes in simulated root resorption defect in MTA group during 4

weeks.

3. To evaluate pH changes in simulates root resorption defect in Portland group during 4

weeks.

4. To evaluate comparative pH changes in simulated root resorption defect in Calcium

Hydroxide group, MTA group and Portland group during 4 weeks.

REVIEW OF LITERATURE

7


Root resorption can lead to progressive destruction of dental hard tissue. Many studies have

reported acidic pH leads to dissolution of mineral component and degrades tissue formation.

The activation of acid phosphatase creates the acidic environment during root resorption

process which is required for demineralization to take place.

Ravi et al: 1988, in a reviewed data on resorption in deciduous teeth and reported that

various inflammatory cytokines that may be responsible for transformation of pre-

odontoclasts to odontoclasts. The pre-existing progenitor cells with proclivity to change into

odontoclasts may cause internal resorption. In primary teeth, loss of protective layer of

predentin over mineralized dentin may also make the tooth more susceptible to resorption.

The cytokines, may lead to transformation of pre-odontoclasts to odontoclasts and loss of

protective layer of pre-dentin.17

Boskey et al:1991, reviewed the data on the role of extracellular matrix components in

dentine during mineralization and reported that extracellular matrix vesicles in the

mineralization process will provide enzymes required for matrix modification. The abundant

proteolytic enzymes present in the vesicles might prepare the matrix for calcification, by

modifying or degrading mineralization inhibitors and then by changing the structure of matrix

components. These enzymes that increase the local phosphate concentration (alkaline

phosphatase, ATPase, etc.) may lead to an increase in the concentration of Calcium and

phosphate in the extracellular matrix fluids. These enzymatic activities would lead to the

precipitation of mineral upon or adjacent to the vesicle. But the activation of acid


8

phosphatase creates the acidic environment during root resorption process which is required

for demineralization to take place.6

Iglesias and jartsfield:2017,reviewed the data on cellular and molecular pathways leading to

external root resorption and explained the (1) adhesion of clastic cell in the external apical

root resorption process and the specific role of extracellular matrix proteins; (2) fusion and

activation by the RANKL/RANK/OPG and ATP-P2RX7-IL1 pathways of clastic cell and

(3)the proteomic and transcriptomic level regulatory mechanisms of root resorption repair by

cementum. In his study he explained that the acidic microenvironment is created during the

process of external root resorption and provides optimal condition for resorption of root.4

The acidic pH environment created during the root resorption can be countered by alkaline

pH which has been reported to be be unfavourable for osteoclastic acid hydrolase activity and

prevent dissolution of mineral component. Several studies have also reported the activation of

alkaline phosphatases by alkaline pH which initiates the hard tissue formation.

The high alkaline pH also exhibits a property of inhibiting residual bacterials including

resistant bacteria such as Enterococcus faecalis, which further stimulates the formation of

hard tissue and remineralization.

De Duve C and Watdaux R:1966, reviewed the data on functions of lysosomal activity and

reported that alkaline pH would be unfavourable for osteoclastic acid hydrolase activity.16


9

Andreasen et al: 1971, reveiwed data on the treatment of fractured and avulsed teeth and

reported that alkaline pH activates alkaline phosphatases and initiates hard tissue formation.8

Stamos et al:1985, reviewed the data on the pH of local anesthetic/calcium hydroxide

solutions and reported that alkaline pH prevents dissolution of mineral component and also

activate alkaline phosphatases.7

McHugh et al: 2004, conducted an in vitro study pH required to kill Enterococcus faecalis.

He tested the growth of Enterococcus faecalis at 0.5 increments from pH 9.5 to 12. Twelve

culture tubes were used in each particluar group. The growth was measured using turbidity

test, a visual scale, and with spectrophotometer. At 24 h, tubes all with pH 9.5 and 10 showed

growth. At 48 h, all tubes with pH 10.5 tubes showed growth. At 72 h, tubes with pH 11

showed growth. After 7 days, there was positive growth in, five of the remaining pH 11

tubes. No growth occurred in any of the pH 11.5 or tubes with pH 12. Hence tubes with pH

10.5 to 11.0 retards growth of E. faecalis, whereas no tubes showed growth at pH 11.5 or

greater thus he concluded that alkaline pH can inhibit Enterococcus faecalis. at p H11.5. 10

The root resorption can be arrested by proper root canal treatment. Many studies have

supported the use of calcium hydroxide as intracanal medicament for treatment of external

root resorption. Calcium hydroxide diffuses the hydroxyl ions which reach the PDL and bone

leading to increased pH values. The high alkaline pH of the calcium hydroxide has the

antibacterial activity and also the ability of inhibiting osteoclastic activity thus creating a


10

favorable environment for formation of hard tissue and preventing the dissolution of

mineralized components. It has also been reported that it also promotes the activating of

alkaline phosphatase which is important for hard tissue formation.

Nerwich et al: 1993, did a study to evaluate the pH changes in root dentin for over a 4-week

period followed by root canal dressing with calcium hydroxide. In this study Root canals of

extracted human teeth were cleaned and shaped and subsequently calcium hydroxide root

canal dressing was given. pH changes in the root dentin were measured over a 4-week period

with microelectrodes in inner and outer dentin at small cavities at apical and cervical levels.

The pH increased within hours in the inner dentin, reaching at pH 10.8 cervically and 9.7

apically. However, from day 1 to 7 before the pH began to rise in the outer root dentin, and

cervically reaching peak levels of pH 9.3 and after 2 to 3 week 9.0 apically. In this study he

reported that when calcium hydroxide is used as a root canal medicament, it releases

hydroxyl ion which diffuses through the dentinal tubules and cementum to reach the PDL.

However if due to trauma or surface resorption, cementum has been removed, then diffusion

of the hydroxyl ions will be faster and more hydroxyl ions will reach the PDL and bone. The

pH in the outer dentine can reach levels of approximately 8.0– 9.5.18

Lengheden et al:1994, conducted a study to evaluate the influence of pH and calcium on

growth and attachment of human fibroblasts. In this study at fixed pH levels ranging between

7.2 and 8.4, human embryonic diploid lung fibroblasts and periodontal ligament fibroblasts

were cultured in media at fixed calcium concentrations ranging between < or = 100 microM

and 20 mM to replicate the effects of calcium hydroxide on vital cell functions such as

attachment and growth. PDL fibroblasts appeared to be more susceptible to changes in pH

and calcium concentration than HEDL fibroblasts. The attachment and growth decreased


11

significantly at pH levels above 7.8. The growth pattern was influenced by changes in pH and

calcium concentration than with attachment. The results explains why intracanal application

of calcium hydroxide through its high pH may impair periodontal healing in areas on the root

surface where the cementum has already been damaged through trauma or periodontal

treatment either, thus the medicament access into the root surface. Hence, the calcium

hydroxide diffused hydroxyl ions through dentinal tubules and it can inhibit osteoclastic cells

to arrest inflammatory resorption, the loss of the protective cementum layer in the region of

the resorption.19

Siqueira et al:1998, conducted a study influence of different vehicles on the antibacterial

effects of calcium hydroxide. In this study influence of three different vehicles on the

antibacterial activity of calcium hydroxide against four bacterial species commonly found in

endodontic infections was evaluated. A broth dilution test was performed using 24-well cell

culture plates. Results showed that all pastes were effective in killing the bacteria tested, but

at different times. The calcium hydroxide/camphorated paramonochlorophenol/glycerin paste

was the most effective against the four bacterial strains tested and explained that the

therapeutic effectiveness of calcium hydroxide dressing materials is based on the release of

hydroxyl ions causing an increase in pH and concluded that Calcium hydroxide exerts

antibacterial effects in the root canal as long as it retains a very high pH.20

Pérez et al:2001, conducted a study on effects of calcium hydroxide form and placement on

root dentine pH. In this study he took extracted single-rooted human teeth were prepared and

instrumented using a conventional technique. Three cavities were drilled at cervical, middle


12

and apical thirds through the root dentine to within 1 mm of the canal wall. A total of 125

teeth were randomly divided into five groups; group 1: aqueous calcium hydroxide paste was

placed in the root canal; group 2: aqueous calcium hydroxide paste was placed in the pulp

chamber; group 3: Hycal, was placed in the pulp chamber; group 4: calcium hydroxide gutta-

percha points were placed in the root canal; group 5: control group, wet canal (distilled water)

without medication. The access cavities and apical ends were sealed, and the teeth were

placed in individual vials containing phosphate-buffered saline, and stored at 37 degrees C.

The pH was measured in the dentinal cavities at 8 h and at 1, 2, 3, 7, 14, and 21 days using a

calibrated microelectrode and reported that calcium hydroxide has its long-term efficacy due

to its property of destruction of bacterial cytoplasm membranes by the liberation of hydroxyl

ions, and the activation of tissue enzymes like alkaline phosphatase.21

Revathi et al:2014, reported in her reviewed data that the alkaline pH of calcium hydroxide

neutralizes the acidic environment in the region of resorption and stimulates hard tissue

formation. The diffusion of hydroxyl ions released by calcium hydroxide would increase the

pH of periodontal space from 6.0 to 7.4 - 9.6.22

Rohit et al:2017, reviewed the data on use of calcium hydroxide in dentistry and reported

that Calcium hydroxide has an alkaline pH which reduces osteoclast activity and stimulates

repair. Calcium hydroxide releases hydroxyl ions and diffusion of hydroxyl ions takes place

through the dentinal tubules that directly communicates with periodontal space and makes the

pH of periodontal space alkaline upto 7.4 - 9.6.23


13

Mineral Trioxide Aggregate (MTA) has been used for vital pulp therapy, root end filling,

apexification and perforation repairs. It has been reported that MTA activates cementoblasts

matrix formation due to its high pH values and its property of releasing substances that

activate cementoblasts. It also exhibits a property of inhibition of microorganisms including

resistant bacterias like Enterococcus faecalis .MTA has also been reported biocompatible,

good sealing property and regenerating properties of tissues such as periodontal ligament and

cemntum.

Torabinejad et al:1995, conducted a study to evaluate the bacterial leakage of mineral

trioxide aggregate as a root-end filling material. In this study Fifty-six single-rooted extracted

human teeth were cleaned and shaped using a step-back technique. Following root-end

resection, 48 root-end cavities were filled with amalgam, Super-EBA, IRM, or MTA. Four

root-end cavities were filled with thermoplasticized gutta-percha without a root canal sealer,

and another four were filled with sticky wax covered with two layers of nail polish. The teeth

were attached to plastic caps of 12-ml plastic vials and the root ends were placed into phenol

red broth, then set-ups were sterilized overnight with ethylene dioxide gas. In 46 teeth (40

experimental, 3 positive, and 3 negative control groups), a tenth of a microliter of broth

containing S. epidermidis was placed into the root canal. In addition, the root canals of two

teeth with test root-end filling materials and one tooth from the positive and negative control

groups were filled with sterile saline. The time required for the test bacteria to penetrate

various root-end filling materials was determined. The samples whose apical 3 mm were

filled with amalgam, Super-EBA, or IRM began leaking at 6 to 57 days. In contrast, the

majority of samples whose root ends were filled with MTA did not show any leakage


14

throughout the experimental duration in this study. In this study he explained that MTA has it

has better sealing ability, high pH, and by releasing substances that activate cementoblasts.24

Mohammadi et al:2006, reviewed data on the Sealing ability of MTA and a new root filling

material and reported that mineral trioxide aggregate (MTA) is a reliable material due to its

biocompatibility, good sealing property, and it encourages regeneration of peri-radicular

tissues such as periodontal ligament bone and cementum.25

Tanomaru et al:2007, conducted a study on in-vitro antimicrobial activity of endodontic

sealers, MTA-based cements and Portland cement and reported that

antibacterial/antimicrobial activity of MTA seems to be associated with elevated pH. He

observed an initial pH of 10.2 for MTA rising to 12.5 in 3 hours and it is known that pH level

in order of 12.0 can inhibit most microorganisms including resistant bacteria such

as Enterococcus faecalis.26

Parirokh et al:2010, reviewed data on Mineral trioxide aggregate clinical applications,

drawbacks, and mechanism of action and reported that MTA is a bioactive material has a

ability to form an apatite like layer on its surface when it comes in contact with physiologic

fluids in vivo or with stimulated body fluid in vitro and MTA can conduct and induct hard

tissue formation.27


15

Cehreli et al:2011, conducted a study on MTA apical plugs in the treatment of traumatized

immature teeth with large periapical lesions. In this case report he described the management

of a late-referral case of periapically involved, traumatized immature permanent incisors by

endodontic treatment and the use of mineral trioxide aggregate (MTA) apical plugs. A 10-

year-old boy with a chief complaint of pain in his maxillary central incisors and history of

subluxation trauma 2 years earlier. Periapical radiograph showed incomplete root

development with wide-open apices and large periradicular lesions. The canals were debrided

using K-files and irrigated with 2.5% NaOCl and 2% chlorhexidine for final flush. The root

canals became asymptomatic after employing the same endodontic regimen for three visits.

In the apical region of the root canals MTA plug were placed, and the rest of the canal space

was obturated by warm compaction of gutta-percha and AH Plus sealer. After 2 months of

treatment the resolution of the large periapical lesions was observed. After 18 months, the

periapical areas showed radiographical evidence of bone healing. After successful removal of

the toxic content of the root canal and placement of MTA plugs resulted in both healing of

the periradicular region and regeneration of the periapical tissue. In this case report he

explained that the production of bone morphogenic protein-2 and transferring growth factor

beta-1 could be two important contributors to the favorable biologic response stimulated by

MTA in periapical tissues. It is also shown that the stimulation of interleukin production by

MTA may influence the over growth of cementum and facilitates the regeneration of

periodontal ligament and formation of bone.28

Hashiguchi et al:2011, conducted a study on Mineral Trioxide Aggregate Inhibits

Osteoclastic Bone Resorption inhibition of cathepsin K and mmp-9 mRNA expression after


16

treatment with MTA solution. He aimed to examine the effect of MTA solution in the

regulation of osteoclast bone-resorbing activity using osteoclasts formed in co-cultures of

primary osteoblasts and bone marrow cells. Dose dependently the MTA solution showed

reduction of the total area of pits formed by osteoclasts. By 20% MTA treatment the

reduction of resorption was due to inhibition of osteoclastic bone-resorbing activity and had

no effect on osteoclast number. A 20% MTA solution disrupted actin ring formation, a

marker of osteoclastic bone resorption, by reducing phosphorylation and kinase activity of c-

Src, and mRNA expressions of cathepsin K and mmp-9. A high concentration of MTA

solution (50%) induced apoptosis of osteoclasts by increasing the expression of Bim, a

member of the BH3-only (Bcl-2 homology) family of pro-apoptotic proteins. In this study he

concluded that MTA is a useful retrofilling material for several clinical situations because it

both stimulates osteoblast differentiation and inhibits bone resorption and the MTA solution

inhibited c-Src-dependent actin ring formation and also the degradation of bone matrix

proteins by suppressing cathepsin K and MMP-9 expression.29

Rahimi et al:2012, conducted a study to evaluate the Osseous reaction to implantation of two

endodontic cements: mineral trioxide aggregate (MTA) and calcium enriched mixture (CEM)

Sixty-three rats were selected and divided into three groups of 21 each. In each femoral bone

the implantation cavities were prepared and randomly filled with the biomaterials only in the

experimental groups. The animals were sacrificed in three groups at 1, 4, and 8 weeks

postoperatively. Histological evaluations comprising inflammation severity and new bone

formation were blindly made on H&E-stained decalcified 6-µm sections. In this study he

reported that MTA filling implantation cavities studied in a rat femur model showed a


17

decrease of the number of inflammatory cells together with the increase of the new bone

formation with the implantation time.30

Saghiri et al: 2014, conducted a study on effect of endodontic cement on bone mineral

density using serial dual-energy X-ray absorptiometry. In this study 40 mature male rabbits

were anesthetized, and a bone defect measuring 7 × 1 × 1 mm was created on the

semimandible. The sample was divided into 2 groups and subdivided into 5 subgroups with 4

samples each based on the defect filled by: Nano-WMTA , WMTA (standard), WMTA

without C3A, Nano-WMTA + 2% Nano-C3A and control group. Twenty and forty days

postoperatively, the animals were sacrificed, and the semimandibles were removed for DXA

measurement and reported that the bone healing and minimal inflammatory response were

obsevered at 3-12 week adjacent to MTAs implanted in proximal rabbit femur.31

Gandolfi MG et al: 2017, conducted a study on osteoinductive potential and bone-bonding

ability of ProRoot MTA, MTA Plus and Biodentine in rabbit intramedullary model. In this

study ProRoot MTA, MTA Plus and Biodentine were used to fill surgical bone in the tibia of

mature male rabbits. Tibiae were retrieved after 30days and submitted to histological analysis

and microchemical characterization using Optical Microscopy and Environmental Scanning

Electron Microscopy with Energy Dispersive X-ray analysis. Bone neoformation and

histomorphometric evaluations, degree of mineralization and the diffusion of material

elements were studied and reported that the MTA allows osteoid matrix deposition by

activating osteoblasts and favours its biomineralization.32


18

MTA’s chief ingredient is Portland cement. Several studies have analyzed the sample of

MTA and Portland cement and reported significant similarity among them. Portland Cement

has been reported biocompatible with similar cellular reactions in comparison to MTA. The

Portland Cement has also shown similar antibacterial activities as of MTA. Portland Cement

exhibits bone healing promoting factors.

Wucherpfenning et al:1999, reviewed data on Mineral trioxide vs Portland cement: two

biocompatible filling materials and reported that MTA consists of Portland cement and

bismuth oxide as a radiopacifier.33

Estrela et al:2000, conducted a study to evaluate antimicrobial and chemical study of MTA,

Portland cement, calcium hydroxide paste, Sealapex and Dycal. The chemical elements of

MTA and Portland cement were analyzed. Four standard bacterial strains: Staphylococcus

aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Bacillus subtilis, one wild fungus,

Candida albicans, and combination of all bacterias were used. 20 ml of BHI agar were

inoculated each in thirty Petri plates with 0.1 ml of the experimental suspensions. Three

cavities, each 4 mm in depth and 4 mm in diameter, were made in each agar plate using a

copper coil and then completely filled with the product to be tested. The plates were pre-

incubated for 1 h at environmental temperature followed by incubation at 37 degrees C for 48

h. The diameters of the zones of microbial inhibition were then measured. Samples were

extracted from diffusion and inhibition halos from each plate and immersed in 7 ml BHI

broth and incubated at 37 degrees C for 48 h. Analyses of chemical elements present in MTA


19

and Portland cement were performed under fluorescence spectrometer. The results showed

that the antimicrobial activity of Calcium hydroxide paste was superior then MTA, Portland

cement, Sealapex and Dycal, as tested from all microorganisms which presented the

inhibitory zones of 6-9.5 mm and diffusion zones of 10-18 mm and reported that the Portland

cement has antimicrobial activities.34

Holland et al:2001, conducted a study to evaluate the healing process after pulpotomy and

pulp covering with mineral trioxide aggregate or Portland cement of dog’s pulp. In this study

after pulpotomy, the pulp stumps of 26 roots of dog teeth were protected with MTA or PC.

After treatment, the animal was sacrificed after 60 days and the specimens were removed and

prepared for histomorphological analysis. A complete tubular hard tissue bridge in almost all

specimens was observed. In conclusion, MTA and PC show similar comparative results

therefore Portland cement can act as a possible substitute for mineral trioxide aggregate.35

Abdullah et al:2002, reviewed the data on Portland cement as a restorative material and

reported that the Portland cement is biocompatible and may have a bone healing promoting

factor.15

Saidon et al:2003, analysed the cell and tissue reactions between mineral trioxide aggregate

and Portland cement. In this study millipore culture plate inserts with freshly mixed or set

material were placed into the culture plates with already attached L929 cells. After an

incubation period of 3 days, the cell morphology and cell counts were studied. Adult male


20

guinea pigs under strict asepsis were anesthetized, during which a submandibular incision

was made to expose the symphysis of the mandible. Bilaterally the bone cavities were

prepared and Teflon applicators were inserted with freshly mixed materials into the bone

cavities. Each animal received 2 implants, one filled with ProRoot and 1 with PC. The

animals were killed after 2 or 12 weeks, and the tissues were processed for histologic

evaluation by means of light microscopy and reported that the cytotoxicity and tissue

reactions of MTA and Portland cement has no difference in cellular reactions.14

De-Deus G et al:2007, in a case report evaluated the use of white Portland cement as an

apical plug in a tooth with a necrotic pulp and wide-open apex and reported that white

Portland cement can be successfully used wide open apex cases with necrotic pulp.36

Conti et al:2009, in a case report evaluated the pulpotomies with Portland cement in human

primary molars. Two clinical cases in which after pulpotomy of mandibular primary molars

in children, the Portland cement was applied presented. Pulpotomy was carried out using PC

in two mandibular first molars and one mandibular second molar, which were further

followed-up. At the 3, 6 and 12-month follow-up appointments of the pulpotomized teeth the

clinical and radiographic examinations revealed that the treatments were successful in

maintaining the teeth asymptomatic and preserving pulpal vitality. Additionally, the

formation of a dentin bridge immediately below the PC was observed and reported that

Portland cement can be effective alternative to MTA.37


21

Sakai et al: 2009, conducted a study to evaluate the pulpotomy of human primary molars

with MTA and Portland cement. In this study thirty carious primary mandibular molars of

children with age group 5-9 years were randomly divided into MTA or PC groups, and treated

with a conventional pulpotomy technique. Then the teeth were restored with resin modified glass

ionomer cement. Clinical and radiographic successes and failures were recorded at 6, 12, 18 and

24-month follow-up and concluded that Portland cement may serve as an effective and less

expensive MTA substitute in primary molar pulpotomies.38

Zeferino et al:2010, conducted a study to evaluate the genotoxicity and cytotoxicity in

murine fibroblasts exposed to white MTA or white Portland cement with 15% bismuth oxide.

In this study at 37°C the aliquots of 1 × 10(4) murine fibroblasts were incubated for 3 h with

MTA (white) or white Portland cement with 15% bismuth oxide, at final concentrations

ranging from 10 to 1000 μg mL(-1) individually and he reported that Portland cement shows

similar genotoxicity and cytotoxicity in comparision to MTA.39

Khalil et al: 2012, conducted a study to evaluate the biocompatibility assessment of modified

Portland cement in comparison with MTA. For comparative in vitro study (MTS test) of the

toxic effect of MTA and MPC with culture isolated from the calvaria of 18-day-old fetal

Swiss OF1 mice was done. A comparative in vivo study in six white New Zealand rabbits for

the biocompatibility of MTA® and MPC was conducted under general anaesthesia. Three

holes (2.5 mm) were made in both the left and right femur. In the first hole MPC was placed,

in the second MTA® and the third one was left empty for negative control group. Three


22

weeks after implantation, two rabbits were sacrificed, then after six weeks two other rabbits

were sacrificed and the last two after twelve weeks. The neck of the femur was trimmed and

prepared for calcified histological studies and reported that MTA and Portland cement

implanted in rabbit mandible showed bone healing and regeneration both in vitro and in

vivo.40

A variety of methods have been used to measure the hydroxyl ion diffusion through dentine.

The initial attempts of pH measurement included use of pH indicating papers and solutions.

These methods had limited accuracy and difficult to interpret. Initial pH measurement means

were reported to give a range of pH values, thus the exact differences in pH could not

interpreted. The newer methods evaluate the pH by electronic pH meters which are reported

to be more user friendly accurate methods with numerical records.

Tronstad et al: 1981, conducted a study to evaluate the pH changes in dental tissues after

root canal filling with calcium hydroxide by using colorimeter which was rather difficult as

the pH indicators changed colors over a range of pH values.9

Gunnar et al:1982, conducted a study to evaluate the pH changes in calcium hydroxide-

covered dentin by using pH indicators with indicator solutions and reported that the pH

indicator gives a range of the pH, thus differences in pH that may exist could not be revealed

by this method.41


23

Huang et al: 1998, conducted a study to evaluate the pH Measurement of Root Canal Sealers

by using pH meter and reported that the determination of pH through pH meter is quite easy

and accurate.42

Larsen et al: 2000, conducted a laboratory study evaluating the release of hydroxyl ions

from various calcium hydroxide products in narrow root canal-like tubes using digital pH

meter and reported it as the most accurate approach with numerical records.43

MATERIALS AND METHOD

24


SOURCE OF DATA

100 extracted teeth (Maxillary central incisors and Mandibular pre-molars) from Dept of Oral

and maxillofacial surgery, Al-Badar Rural Dental College, Gulbarga, Karnataka

INCLUSION CRITERIA:

1. Single rooted, mature teeth with intact crown.

EXCLUSION CRITERIA:

1. Teeth which had dental caries, attrition, abrasion, cracks or fractures.

ARMAMENTARIUM AND EQUIPMENTS:

1. Radiographs

2. Saline

3.Diamond disk

4. Pro-taper files


25

5. Irrigation needle

6.Sodium hypochloride

7. Ethylene diamine tetra acetic acid(EDTA)

8. Distilled water

9. Round bur No 2

10.Airoter

11.Paper towel

12.Paper points

13.Hand pluggers

14.Zinc oxide eugenol(ZnO eugenol)

15.Sticky wax

16.Sintillation vial

17.pH meter

18.MTA carver

MATERIALS:

1. Calcium hydroxide

2. Mineral trioxide aggregate


26

3. Portland cement

METHOD

100 extracted teeth, which fulfilled the selection criteria were collected and stored in saline

until use and between preparation manipulation. Radiographs (mesial and distal angulations)

were used to confirm the presence of a single canal. Teeth were soaked in sodium

hypochloride for 30 minutes, gently scaled to remove organic debris, taking care to avoid

damage to cementum and rinsed in distilled water. Decoronation with a standard length of 14

mm was performed with a diamond disk. Size 10 K file was used until a loose and smooth

glide path was confirmed, considering 14 mm as working length. The same length was

transferred to Protaper S1 and S2 files. The secured portion of the canal was optimally pre-

enlarged by first utilizing S1 then S2 for preparing coronal two third of the canal .The canals

were irrigated with 3 ml of 3% NaOCl for 30s and recapitulated by size 10 K file, followed

by use of each shaping file. The apical one-third of the canal was enlarged to at least a size 15

K file upto working length. For shaping apical one-third Protaper F1, F2, F3 was used

followed by size 20, 25, 30 K file respectively. The canals were irrigated with 3 ml of 3%

NaOCl after using each instrument. After instrumentation, canals were irrigated with 3 ml of

17% EDTA for 3 minutes, followed by 3 ml of 3%NaOCl and lastly with 3 ml distilled

water.

To simulate root resorption defect on the buccal surface of root, a cavity preparation was

done at 5 mm from apex(1.2 mm diameter and 0.6 mm deep) by using a 1.2 mm carbide

round


27

bur. Root surface cavity was rinsed with 3 ml 17% EDTA, for 1 min and followed by 3 ml

distilled water.

Placement of Ca(OH)2, MTA and Portland cement

The teeth were randomly equally divided into 4 groups

Group 1:- Ca(OH)2 (n=30 teeth)

Group 2 :- MTA (n= 30 teeth)

Group 3 :- Portland cement(n=30 teeth)

Group 4 :- Control group

Saline(n=10 teeth)

Prior to the beginning of the study, teeth were removed from storage medium and blotted dry

with paper towel, root canals were dried with sterile paper points. Root canals of group I, II,

III were filled with Ca(OH)2, MTA, Portland cement and control group with saline upto

12mm from apex. Successful placement was evaluated with radiographs. Coronal access

opening was sealed with ZnO eugenol cement. The apical 3 mm was covered with sticky wax

to seal the foramen .The coronal part was attached to the internal surface of a scintillation vial

lid by sticky wax and lid will placed on vials filled with saline.


28

pH measurement

After every 20 min, 3 hours, 24 hours, 1 week, 2 weeks, 3 weeks, 4 weeks each tooth was

removed from vial by unscrewing the lid, rinsed with distilled water and briefly dried with

paper towel. The root surface was filled with distilled water and left for 3 min after which the

pH was noted with calibirated digital pH meter.

SAMPLE SIZE ESTIMATION

29

Sample size estimation

Analysis: Compromise: Compute implied α & power

Input: Effect size f = 0.5

β/α ratio = 0.5

Total sample size = 90

Numerator df = 10

Number of groups = 3

Number of covariates = 1

Output: Noncentrality parameter λ = 11.2500000

Critical F = 1.2494900

Denominator df = 41

α err prob = 0.2902175

β err prob = 0.1451087

Minimum Total Sample size is 90 (30 in each group) for a power of 0.95

OBSERVATION AND RESULTS

30


Statistical analysis was done by one way ANOVA and Tukey test to evaluate the changes in

pH over time in control and experimental (Ca(OH)2, MTA, Portland cement)groups in

simulated root cavities. The analysis was done using Minitab19 software.


31

OBSERVATION-

The mean values for the 3 experimental groups and control groups at each time point are

presented in Table 1.

20 min

3

hr 24 hr 1 wk 2 wk

3

wk 4 wk Total

Ca(OH)2

Count 30 30 30 30 30 30 30 210

Sum 272.64 277.9 276.41 261.08 244.45 242.68 240.13 1815.29

Average 9.088 9.2633 9.2136 8.7026 8.1483 8.0893 8.0043 8.6442

Variance 0.0067752 0.0367 0.0050 0.0011 0.0406 0.0856 0.0425 0.2989

MTA

Count 30 30 30 30 30 30 30 210

Sum 272.06 281.05 278.21 265.63 251.23 247.09 247.66 1842.93

Average 9.06866 9.3683 9.2736 8.8543 8.3743 8.2363 8.2553 8.7758

Variance 0.00678 0.0716 0.0903 0.0344 0.0145 0.0268 0.0228 0.240

Portland

Count 30 30 30 30 30 30 30 210

Sum 272 280.89 276.33 265.22 253.09 249.66 250.26 1847.45

Average 9.0666 9.363 9.211 8.8406 8.4363 8.322 8.34 8.7973

Variance 0.0019 0.0204 0.0322 0.0344 0.0132 0.0138 0.0258 0.1817

Control

count 10 10 10 10 10 10 10 70

sum 73.45 73.3 73.3 73.29 73.27 73.19 73.17 512.97

average 7.34 7.33 7.33 7.32 7.32 7.319 7.317 7.32

variance 0.0012 0.0002 0.004 0.0008 0.0007 0.0002 0.0004 0.0001

Table 1-Summary of pH values of each experimental and control group.


32

RESULTS-

Intragroup effects-

CALCIUM HYDROXIDE

The pH changes occurred over 4 week experimental period within Ca(OH)2 group are

significant. The pH increased between 20min to 3 hr. The pH decreased between 3hr to 24hr,

1week, 2 week, 3 week, 4 week.

Graph 1-Changes in pH of Calcium Hydroxide during 4 weeks

7

7.5

8

8.5

9

9.5

20 min 3 hours 24 hours 1 week 2 week 3 week 4 week

calcium hydroxide


33

MINERAL TRIOXIDE AGGREGATE

The pH changes occurred over 4 week experimental period within MTA group is significant.

The pH increased between 20min to 3 hr. The pH decreased between 3hr to 24hr, 1week, 2

week, 3 week, 4 week.

Graph 2-Changes in pH of MTA during 4 weeks

7

7.5

8

8.5

9

9.5


MTA


34

PORTLAND CEMENT

The pH changes occurred over 4 week experimental period within Portland cement group is

significant. The pH increased between 20min and 3 hr. The pH decreased between 3 hr to 24

hr, 1week, 2 week, 3 week, 4 week. However, the pH changes in the Portland cement group

over 2 weeks was higher in comparison to other experimental groups namely Ca(OH)2 and

MTA.

Graph 3-Changes in pH of Portland Cement during 4 weeks

7

7.5

8

8.5

9

9.5


portland cement


35

CONTROL

The pH readings for control did not differ significantly during 4 week experimental period

Graph 4- Changes in pH of Control group during 4 weeks

7.2

7.25

7.3

7.35

7.4

7.45

7.5

20 min 3 hr 24 hr 1 week 2 week 3 week 4 week

control


36

Intergroup effects-

Comparative pH changes occurred over 4 week experimental period among the experimental

groups are as follows:

1. The pH changes that occurred over 4 week within Ca(OH)2 and MTA groups are

significant. The MTA group showed overall mean pH higher at the end of 4 week

experimental period in comparison to Ca(OH)2 group. The MTA group also showed

higher pH in comparison to Ca(OH)2 group at all intervals of experimental period i.e,

after 20 min, 3 hr, 24 hr, 1 week, 2 week, 3 week, 4 week.

2. The pH changes occurred that over 4 week within Ca(OH)2 and Portland cement

groups are significant. The Portland cement showed the overall mean pH higher at

the end of 4 week experimental period in comparison to Ca(OH)2 group. The Portland

cement group also showed higher pH in comparison to Ca(OH)2 group at all intervals

of experimental period i.e, after 20 min, 3 hr, 24 hr, 1 week, 2 week, 3 week, 4 week.

3. The pH changes that occurred over 4 week within MTA and Portland cement groups

are insignificant. The Portland cement group showed the overall mean pH higher at

the end of 4 week experimental period in comparison to MTA group. The Portland

cement group also showed higher pH in comparison to MTA group at the interval of 2

week, 3 week and 4 week.


37

Graph 5-Comparision of changes in pH among Ca(OH)2, MTA, Portland Cement during 4

weeks

7

7.5

8

8.5

9

9.5

20 min 3 hr 24 hr 1 wk 2 wk 3 wk 4 wk

Ca(OH)

MTA

Portland

DISCUSSION

38

DISCUSSION

Root resorption can lead to either a physiological or pathological process ending in a loss of

tissue such as dentin, cementum and alveolar bone. Physiological root resorption associated

with primary teeth is desirable because it results in exfoliation of the primary teeth helping in

eruption of the permanent teeth. However, root resorption of permanent dentition is

undesirable.

Hard tissues are protected by surface layers of blast cells. The longer these layers are intact,

resorption cannot occur. The bone, dentine and cementum are mesenchymal tissues which are

composed of collagen and hydroxyapatite crystals, though their susceptibility to resorption

markedly differs. Root resorption might be classified by its location that is, internal or

external resorption. Internal root resorption is initiated within pulp tissue whereas external

root resorption starts in the periodontal ligament and is classified by location as apical, lateral

or cervical.

Internal resorption is a rare occurrence in permanent teeth. Internal root resorption has an

uncommon occurrence and its etiology is also poorly understood. The predisposing factors

for internal root resorption can be trauma, pulpitis, pulpotomy, cracked tooth, tooth

transplantation, restorative procedures, orthodontic treatment or Herpes zoster viral infection.

On Radiographic examination, it can be characterised by an oval shaped enlargement in the

root canal space. Histologically the internal root resorption can be characterized by

multinucleated giant cells adjacent to granulation tissue in the pulp.

DISCUSSION

39

The etiology behind the external root resorption of permanent teeth is usually the result of

trauma, chronic inflammation of pulp/periodontal tissues or both, induced pressure in

periodontal ligament (orthodontic movement), tumours or tooth eruption.2

The different forms of external root resorption have been described in literature. For some of

these the underlying mechanism is understood but some other forms are still unexplained and

they are therefore, termed as idiopathic.

A classification system for external root resorption with known mechanism is as follows:

Surface resorption

Replacement resorption associated with ankylosis

Inflammatory resorption

An injury to cementoblastic layer subsequently initiates surface resorption. The denuded root

surface attracts the cementoclast cells, which resorbs the cementum. As the resorption stops,

cells from the surrounding periodontal ligament tissue proliferates in the resorbed area and

results in deposition of new reparative dental tissue.44-45In case of minor trauma caused due to

unintentional biting on hard subjects or bruxism the localized damage to the periodontal

ligament tissue can trigger this type of resorption. But this process is self-limiting and can be

reversible.

Replacement resorption results in replacement of bone in the dental hard tissue. When a

surface resorption stops, the cells will proliferate from the periodontal ligament in the

resorbed area. If the resorption area is large, the cells from the nearby bone may start arriving

DISCUSSION

40

first and start to establish them on the resorbed surface.46 Thus the bone will be formed

directly upon the dental hard tissue resulting in fusion between bone and tooth also known as

ankylosis.

Apical periodontitis can cause apical root resorption also known as inflammatory root

resorption. There are two main forms of external resorption associated with inflammation in

the periodontal tissues

Peripheral inflammatory root resorption

External inflammatory root resorption

Both of them are triggered by destruction of the blast cells in the adjacent tissues. In

Peripheral inflammatory root resorption, the inflammatory lesion in the adjacent periodontal

tissues trigger the osteoclast activating factors, initiates the resorptive process.47,48 This type

of resorption is commonly situated at cervical regions, immediately apical to the marginal

tissues and often termed as cervical root resorption. But the location is not always cervical in

position as it is related to the level of the marginal tissues and the pocket depth.

The external inflammatory root resorption gets initiated from an infected necrotic pulp.

Followed by dental trauma due to damage of the periodontal ligament the resorptive process

begins as a surface resorption. The pulp also gets damaged and latter becomes necrotic. As

the surface resorption reaches to the dentine, the osteoclasts carry the resorptive activity. The

necrotic and the infected pulp matter gets released to the exposed dentine tubules.47,48 The

infected pulp products maintain the inflammatory process in the adjacent tissues which

continues the resorptive process.

DISCUSSION

41

In a study by Heinz et al 2015 the periodontal ligament and bone marrow derived circulating

mononuclear hematopoietic precursor cells directs odontoclastic differentiation. The cell

commitment and mononuclear cell fusion are regulated by E- cadherin which is required for

cell to cell adhesion. Activated osteoclastic cells get attached to mineral matrix, forming a

sealing zone and adopting a polarized morphology which contains ruffled border and secrete

proteases which initiate mineral resorption. V-ATPase pump carries the protons produced by

carbonic anhydrase II to these ruffled border membrane and releases them into the resorption

pit and generate an acidic microenvironment which is completed by chloride transport. TRAP

enzymes are responsible for endocytosed material elimination. Finally, the end result of

resorption process are degradation of the organic component.4 The MMPs and cathepsins are

responsible for degradation of collagen rich organic bone matrix.

The optimal condition for resorption to take place is an acidic pH. At an acidic pH, the acid

hydrolases gets active leading to demineralization. Several studies has reported that the

alkaline pH would be unfavorable for osteoclastic acid hydrolase activity.16 At an alkaline pH

dissolution of mineral component is prevented and it also activate alkaline phosphatases,

which is responsible for the hard tissue formation.7,8

The aim of endodontics is preservation the natural teeth. Hence a number of medicaments

have been introduced. One of those agents is calcium hydroxide. Calcium hydroxide was first

introduced by Hermann in 1920, Germany. Since then it has been introduced for numerous

purpose such as pulp capping, apexogenesis, apexification, root perforations, root fractures,

replantation, intercanal dressings and root resorptions. There are several theories about the

mechanism of action of calcium hydroxide. According to Rehman et al calcium hydroxide

dissociates into calcium and hydroxyl ions. The hydroxyl ions are

DISCUSSION

42

responsible for the alkaline pH and for the bactericidal properties.

Several studies have been conducted on diffusion of calcium and hydroxyl ions. Wang and

Hume in their in- vitro study observed a slow movement of hydroxyl ions through dentine

and showed dentine’s ability to buffer hydroxyl ions. Tronstad et al in histological sections of

monkey teeth observed in resorptive areas (induced resorption area) reported increased pH

extended to dentinal surface. Fuss et al measured pH changes in distilled water surrounding

teeth filled with calcium hydroxide and observed changes in pH levels. In a similar study

Nerwich et al showed changes in pH level which were higher at cerivical region as compared

to apical region. According to Bystrom et al most endodontic pathogens cannot survive in

calcium hydroxide’s high alkaline pH. In his study he showed the lethal effects of calcium

hydroxide by elimination of several bacterias commonly found in infected root canals.

According to Stamos and Andreasen an alkaline pH prevents dissolution of mineral

component and might also activate alkaline phosphatases, which is important for hard tissue

formation.7,8

Mineral trioxide aggregate (MTA) is a tricalcium mineral complex, introduced by

Mohmoud Torabinejad, USA in 1993. It can be used for perforation repair, retrograde

filling, apexification, vital pulp therapy and root resorption. In combination with water,

calcium ions are released and immediately high alkaline pH is obtained which may last up

to several months. Due to the high pH and ability to stimulate cementoblasts/odontoblasts,

it can be used in cases of root resorption pathologies.49,50Al-Hazaimi et al. (2006) reported

that the MTA has antibacterial activity against Enterococcus faecalis and Streptococcus

DISCUSSION

43

sanguis.51 According to Holland et al. (1999) the MTA interacts with tissue fluids and form

Ca(OH)2 , which results in hard-tissue formation.52 Torabinejad et al. (1995) reported that

MTA has a potential to activate the cementoblasts and eventually the production of

cementum.53 Faraco et al. (2001) reported that the dentinal bridges formed with MTA were

relatively faster and showed better structural integrity in comparision with Ca(OH)2 .54

In several studies an alkaline pH induction was observed in simulated resorptive defects in

extracted teeth filled with Ca(OH)29,18,43,55-57 and alkaline pH induction by MTA.26,58 In

comparative study by Sarah Heward and Christine M reported placement of intracanal MTA

compared with Ca(OH)2 showed higher pH which is consistent with the present observations

of this study. However, this is the first study evaluating the diffusion of hydroxyl ions from

Portland cement by measuring pH in stimulated root surface cavities.

The diffusion of hydroxyl ions through dentinal tubules may get slowed by buffering capacity

of dentin.49 Buffering mechanism occurs when proton donors in hydrated layer of

hydroxapatite provides additional protons to keep pH unchanged.18 Hydroxyl ions may get

absorbed into hydrated layer of hydroxyl apatite crystals leading to further slowing their

diffusion.59,60 Foster et al 1993, conducted a study to evaluate the effects of smear layer

removal on the diffusion of calcium hydroxide through radicular dentin and reported that the

removal of the smear layer facilitated the diffusion of hydroxyl ions through the dentinal

tubules and improved the ability to kill bacteria.61,62 Hence, in this study 17% EDTA

followed by 3% NaOCl for the final irrigation was done to ensure the removal of smear layer

in the internal canal wall prior to the placement of experimental materials, with a purpose of

facilitating hydroxyl ion diffusion through dentin.

DISCUSSION

44

In 1993 Nerwich et al did a study to evaluate the pH changes in root dentin and reported that

hydroxyl ion diffuses through the dentinal tubules and cementum to reach the PDL. If

cementum has been removed by the trauma or by surface resorption, then diffusion of the

hydroxyl ions will be faster and more hydroxyl ions will reach the PDL and bone.18 In a

resorptive lesion the root surface is damaged by activated odontoclasts and the cementum

gets denuded. In this study the simulated root surface cavities were prepared on the root

surface to experimentally induce pathologically resorptive lesion.

Hammarstrom et al and later Lengheden et al showed in stimulated root cavities initially

calcium hydroxide intracanal dressing caused necrosis of osteoclastic cells and postulated

hydroxyl ion diffusion through dentin caused these effects.63,64

There are different methods for intracanal placement of calcium hydroxide as described in

several studies.65-67 Simcock and Hicks evaluated the different methods of efficacy in

minimally and fully prepared canals. They reported that with respect to completely prepared

canals, all delivery methods were efficient.65In the present study calcium hydroxide was

placed by counter clockwise rotated reamer in a completely prepared canal.

In the present study the diffusion of hydroxyl ions during first 24 hr was similar for each of

experimental materials; however there was a steeper decline in pH for Ca(OH)2 in

comparison with MTA and Portland cement. While MTA and Portland cement showed no

significant changes. The increase in pH might be attributed to initial setting time of the

materials. The immersion fluid was not replaced during the study period; however it might be

a cause of equilibration consequent to prior diffusion into a static external solution leading to

decline of pH. Whereas in a similar study Heward and Sedgley 2011 has argued, placing

immersion solution regularly could better simulate the in vivo situation as tissue fluid

circulation in areas of resorption might facilitate diffusion.

DISCUSSION

45

The rationale behind treatment of external root resorption with calcium hydroxide is that the

acidic pH produced by resorptive cells would be neutralized, thus creating an alkaline

environment and preventing dissolution of mineral components. The rise in pH is not

optimum for the activity of osteoclastic acid hydrolases and their activity gets inhibited. An

alkaline pH also activates alkaline phosphates which plays a leading role in remineralization.9

In addition to the neutralizing pH the presence of calcium ions also play an important role in

arresting and healing effects of root resorption. The calcium ions are important for the

activity complement system in an immunologic reaction. Calcium ions also activate calcium

dependent ATP-ase, which are associated with hard tissue formation. 68

Calcium hydroxide also sustains an antibacterial property and shown to detoxify

lipopolysaccharide in vivo thus reduces microbial load and limiting the diffusion of toxins

through dentinal tubules.69 In in-vitro experiments with calcium hydroxide have shown that

when endodontic bacteria were added to its suspension, 26 out of 27 strains were killed in

less than 6 mins.70

The present study showed that during the period of 4 weeks, teeth filled with Ca(OH)2

showed significant pH changes which is in agreement with similar study done by Heward and

Sedgley 2011. Siquera and Uzeda demonstrated in their study the calcium hydroxide was not

able to eliminate the E.faecalis and F. nucleatum. In a study conducted by Safavi et al

showed that even after extended period of calcium hydroxide treatment E.faecalis remain

viable. In several studies Calcium hydroxide has also been reported with disadvantage of

increased risk to fracture of the concerned tooth11 and patient compliance as it needs multiple

appointments71 whereas MTA provides a good alternative to calcium hydroxide.

DISCUSSION

46

MTA is a bioactive material has a ability to form an apatite like layer on its surface when it

comes in contact with physiologic fluids in vivo or with stimulated body fluid in vitro and

MTA can conduct and induct hard tissue formation.27 In several studies MTA was reported to

have a capacity to activate cementoblasts to produce matrix formation as it has better sealing

ability, high pH, and by releasing substances that activate cementoblast and encouraging

regeneration in periradicular tissues such as periodontal ligament, bone and cementum.24-

25,27,30 Gandolfi et al conducted a study on osteoinductive potential and bone-bonding in

rabbit intramedullary mode for Microchemical characterization and histological analysis and

reported that the MTA allows osteoid matrix deposition by activating osteoblasts and favours

its biomineralization.32

In an in-vitro study Tanomaru et al reported that MTA has antibacterial/antimicrobial

activity. He observed an initial pH of 10.2 for MTA rising to 12.5 in 3 hours and it is known

that pH level in order of 12.0 can inhibit most microorganisms including resistant bacteria

such as Enterococcus faecalis.26 In agreement with his study, the present study showed that

during the period of 4 weeks, teeth filled with MTA showed significant pH changes over 3

hours.

MTA and Portland cement are reported to have a significant similarity. MTA consists of

Portland cement and bismuth oxide as a radiopacifier.33 In several studies Portland cement

has been reported as an alternative for MTA.35-38,40 Portland cement has been reported as a

biocompatible material and reported to have a bone healing factor.15,40 Saidon et al 2003

reported that the cytotoxicity and tissue reactions of MTA and Portland cement has no

difference in their cellular reactions.14 Khalil et al. conducted a study to evaluate the

biocompatibility assessment of modified Portland cement in comparison with MTA and

DISCUSSION

47

reported that MTA and Portland cement implanted in rabbit mandible showed bone healing

and regeneration.40 In a vitro study conducted by Estrela et al 2000 reported that Portland

cement consists of antimicrobial activities.34

This study showed that during the period of 4 weeks, teeth filled with Ca(OH)2, MTA and

Portland cement showed significant pH. The overall pH values in root surfaces cavities of

MTA and Portland cement were higher in comparison with Ca(OH)2. The MTA and Portland

cement showed insignificant changes in their comparative pH values in root surface cavities

during the experiment. MTA and Portland cement share the property of similar pH, however,

the Portland cement showed higher pH values in comparison to MTA, during the

experimental duration. In several studies Portland cement has shown no difference in cellular

reactions as compared of MTA. The Portland cement has been proven a biocompatible

material with bone healing and promoting factor. Therefore, due to the effectivity and low

cost of Portland cement and similar properties as compared to MTA, Portland cement can be

considered as a less expensive and equally effective possible substitute and further in vitro

and in vivo studies in this regard with larger sample size are indicated.

CONCLUSION

48

CONCLUSION

The conclusions of this study are-

1) All the experimental groups namely Ca(OH)2 , MTA and Portland cement in root

surface cavities showed significant pH changes during 4 week experimental period

individually.

2) Ca(OH)2 and MTA in root surface cavities showed significant changes in pH after 4

weeks. MTA showed higher alkaline pH in comparison to Ca(OH)2.

3) Ca(OH)2 and Portland cement in root surface cavities showed significant changes in

pH after 4 weeks. Portland cement showed higher alkaline pH in comparison to

Ca(OH)2.

4) The Portland cement showed higher alkaline pH than MTA at the end of 4 week

experimental period. Within the limitation of this study, these results indicate that it

may be efficacious to use Portland cement in root resorption cases and further in vitro

and in vivo studies in this regard with larger sample size are indicated.

SUMMARY

SUMMARY

Root resorption is described as the condition associated with either a physiological or

pathological process resulting in a loss of tissue such as dentin, cementum and alveolar bone.

It has been shown that resorption process may be arrested by proper endodontic therapy.

Traditionally Ca(OH)2 has been used as an intracanal medicament for treatment of external

inflammatory root resorption. The high alkaline pH greater than 12, of Ca(OH)2 has the

ability to not only kill micro-organisms but also neutralize osteoclasts thus preventing

dissolution of the mineral components. MTA has been used for vital pulp therapy, root end

filling, apexification and perforation repairs. It is biocompatible and can perfectly seal dentin.

And as compared with Ca(OH)2 it shares similar property of high alkaline pH and inhibition

of microorganisms. Further Ca(OH)2 has disadvantage of increased risk to fracture. Hence

MTA provides as very good alternative.

MTA’s chief ingredient is Portland Cement. Portland cement shares similar properties as

compared to MTA. Various studies have been done on diffusion of hydroxyl ions from

several intracanal medicaments and MTA has showed significant results with high amount of

alkaline pH. But MTA is expensive. In order to search for other cost effective alternatives

which also possess similar properties, it is necessary to assess the comparative properties of

newer materials. Portland cement is not only the chief constituent of MTA but also known to

be cost effective. The present study aims comparison of intracanal Calcium Hydroxide,

Mineral Trioxide Aggregate and Portland Cement to induce pH changes in simulated root

resorption defects in human teeth. 100 extracted teeth were decoronated with a standard

length of 14 mm. Root canal preparation was performed by using Pro Taper rotary system.

To simulate root resorption defect on the buccal surface of root, a cavity preparation was

done at 5 mm from apex (1.2 mm diameter and 0.6 mm deep). Teeth were randomly equally

SUMMARY

divided and filled with Calcium hydroxide, MTA and Portland cement. Successful placement

was evaluated with radiographs. Subsequently, pH measured after every 20 min, 3 hours, 24

hours, 1 week, 2 weeks, 3 weeks, 4 weeks of each tooth with calibrated digital pH meter.

All the experimental groups namely Ca(OH)2 , MTA and Portland cement in root surface

cavities showed significant pH changes during 4 week experimental period individually.

MTA and Portland cement showed higher alkaline pH changes in comparison to Ca(OH)2.

The Portland cement showed highest alkaline pH changes among all the groups at the end of

4 week experimental period.

The pH changes in root surface cavities of Portland cement were highest in comparison with

MTA and calcium hydroxide during the 4 week experimental period. These results indicate

that it may be efficacious to use Portland cement in root resorption cases.

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49

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PHOTOGRAPHS

F

Figure 1- Teeth specimen

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Figure 2 –Radigraphs for confirmation of single canal (a) parallel angulation (b) mesial

angulation (c) distal angulation

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Figure 3- Decoronated specimen

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Figure 4 Shaping of canals

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Figure 5- Cavity preparation at 5 mm coronal to the apical foramen

Figure 6- Radiographs for confirmation of voidless placement of materials

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Figure 7- Prepared samples in scintillation vial

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Figure 8- Digital pH meter

COMPARISON OF INTRACANAL CALCIUM HYDROXIDE, MINERAL ...

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