“an evaluation of the effect of different irrigating

i

“AN EVALUATION OF THE EFFECT OF DIFFERENT IRRIGATING

SOLUTIONS AS A FINAL RINSE ON THE MICROHARDNESS OF ROOT

CANAL DENTIN - AN IN VITRO STUDY’’

Dissertation Submitted to the Rajiv Gandhi University of

Health Sciences, Karnataka; Bangalore

In partial fulfilment

Of the requirements for the degree of

MASTER OF DENTAL SURGERY

In

CONSERVATIVE DENTISTRY AND ENDODONTICS

Under the guidance of

DR. JAYAKUMAR T., M.D.S, PROFESSOR

DEPARTMENT OF CONSERVATVE DENTISTRY AND ENDODONTICS,

THE OXFORD DENTAL COLLEGE,

BOMMANAHALLI, BANGALORE- 68

2016-2019

By

DR. NOORIE ABDUL SALAM

Scanned by CamScanner

i

List of abbreviations

ANOVA Analysis of Variance

Ca:P Calcium: Phosphate

ClO2 Chlorine Dioxide

EDTA Ethylenediaminetetraacetic acid

ISO International Organisation for Standardisation

K-Files Kerr files

KHN Knoop Hardness Number

NaOCl Sodium Hypochlorite

NiTi Nickel Titanium

SD Standard Deviation

VHN Vicker’s Hardness Number

i

List of tables

Sl. No. Tables Page No.

1. Comparison of mean Micro Hardness values

between 05 groups using One-way ANOVA Test

41

2. Multiple comparison of mean difference in Micro

hardness values between groups using Tukey's HSD

Post hoc Analysis

42

3. Individual values obtained 65

List of graphs

Sl. No. Tables Page No.

1. Comparison of mean Micro Hardness values

between 05 groups

43

2. Comparison of mean Micro Hardness values [with

standard errors] between 05 groups

44

i

List of Photographs

Fig. No. Photographs Page No.

1. Teeth samples 39

2. Protaper Universal rotary files 39

3. Endomotor 39

4. Teeth embedded in resin 39

5. Saline 39

6. Sodium hypochlorite 39

7. EDTA 40

8. Chlorine dioxide 40

9. QMix 40

10. Matricaria recutita 40

11. Application of irrigating solution 40

12. Vicker’s microhardness test 40

13. Illustration of the Vicker’s indentation and

digital measurement of the diagonals

68

i

ABSTRACT

Aim:

The objective of this in vitro study is to evaluate the effect of EDTA, ClO2, QMix,

and Matricaria recutita, on the microhardness of root dentin.

Materials & Methodology:

Forty extracted single rooted premolars were randomly assigned to 5 groups (n=16).

The root canals were prepared using Protaper Universal rotary files (Dentsply, India)

and distilled water for irrigation. Each group was treated with NaOCl as initial rinse

and then with EDTA, ClO2, QMix, Matricaria recutita, and Saline as final rinse. All

the samples were then placed on the Vicker’s microhardness tester and the results

statistically analyzed to determine which irrigating solution had the least effect on

the microhardness of root dentin.

Results:

Matricaria recutita had the least effect on the microhardness of root dentin, followed

by QMix and ClO2. EDTA showed the maximum reduction in microhardness of root

dentin.

Conclusion:

Under the limitations of this study, Matricaria recutita and QMix show promise for

use as irrigants because of less detrimental effect on the microhardness of root

dentin.

Keywords: EDTA, ClO2. German chamomile, QMix, root dentin, irrigation, microhardness, Vicker’s, NaOCl, final rinse .

Introduction

1

A successful root canal treatment involves the root canal system being

thoroughly cleansed and disinfected, followed by a three dimensional obturation of

this space.1 The mechanical instrumentation of the root canal produces a smear layer

that covers the dentinal tubules. The smear layer, being an amorphous irregular layer

containing inorganic dentin debris as well as organic materials like pulp tissue,

odontoblastic process, necrotic debris, microorganisms and their metabolic by-

products, needs to be removed before and during shaping.2 Hence, it involves the

simultaneous action of endodontic instruments and irrigating solutions which

eliminate the pre-existent organic and inorganic remnants and reduce the microbial

content and its by-products from the root canal system.2

Irrigation is one of the most important aspects of root canal preparation and is

currently the best method for the removal of tissue remnants and dentin debris during

instrumentation.3 It facilitates this removal through a flushing mechanism and can

also help in preventing the packing of hard and soft tissue in the apical third of the

root, which could otherwise cause a variety of complications such as transportation,

zipping, and extrusion of infected material into the periapical area. Berutti et al. in

1997 stated that solutions used as irrigants could penetrate 130µm of the dentinal

tubules, but bacteria can penetrate up to 1000µm; and to achieve proper antibacterial

effectiveness, irrigating agents should remove organic and inorganic tissues in root

canals, open the tubules, and penetrate in to the root canal system.4 Because of the

central role of irrigation in endodontic treatment and their multitude of tasks, there is

a continuing search for an optimal irrigating solution and strategy. None of the

presently available irrigating solutions can be regarded as optimal; however, using a

Introduction

2

combination of products in the correct sequence can contribute to a successful

treatment outcome.5

The most widely used endodontic irrigant is sodium hypochlorite (NaOCl),

because of its antibacterial activity and ability to dissolve vital and necrotic organic

tissue.6 It is formed by the bonding of chemical compounds like hypochlorous acid

(HOCl) and Sodium hydroxide (NaOH). It is hypertonic and alkaline which makes its

pH higher than 11, and is antibacterial, acts as a solvent of organic matrix, oxidizes

and hydrolyzes protein, removes intracellular fluids as well as magnesium and

carbonate ions. It also destroys fungi, spores, and viruses when used in low

concentrations ranging from 1% to 5.25%, thereby reducing its toxicity.7

Hypochlorite is the only root canal irrigant of those in general use, that dissolves

necrotic and vital organic tissue. It is difficult to imagine successful irrigation of the

root canal without sodium hypochlorite.5

The complete removal of smear layer requires the use of a chelating agent or

other demineralizing agents and a soft tissue solvent because no single solution can

provide both effects.8 Nygaard Ostby in 1957 introduced chelating agents in

endodontics for the preparation of calcified and narrow canals. Chelation is a physio-

chemical process which involves the uptake of multivalent positive ions, reacts with

the calcium ions in hydroxyapatite crystals and causes changes in the microstructure

of the dentin and changes the Ca:P ratio of the dentin surface.2 EDTA according to

Nygaard Ostby was recommended to be used at 15% concentration with a pH of 7.3

and the following composition: Disodium salt of EDTA (17g), Distilled water

(100mL), 5M sodium hydroxide (9.25mL). The main mineral components of dentin,

Introduction

3

phosphate and calcium, are soluble in water. When the disodium of EDTA is added to

this equilibrium, the calcium ions are removed from the solution which leads to the

further dissolution of the ions from the dentin, keeping the solubility of the product

constant and thus causing decalcification of the dentin.5 Based on a review on the

applications of EDTA in endodontics by Mazen et al. in 2017, EDTA decalcified

dentin to a depth of 20-30µm in 5 minutes. The liquid EDTA at a concentration of

17% removes the smear layer better as compared to paste type EDTA when it comes

in contact with the root canal dentin for less than 1 minute.9 According to a study by

Jose et al. in 1998 which studied the antibacterial properties of endodontic irrigants,

the antimicrobial effect of EDTA was stronger than that of citric acid and 0.5%

NaOCl but weaker than 2.5% NaOCl and 0.2% CHX.10

Chlorine dioxide (ClO2) is chemically similar to chlorine or hypochlorite and

this makes it a likely substitute for the hypochlorite due to its reduced toxicity and

irritating effect when applied to the human body. According to the manufacturer, it is

known to have certain tuberculocidal, bactericidal, virucidal and fungicidal properties

and is known to kill bacteria by disrupting the transport of nutrients across the cell

wall, due to its powerful oxidizing properties. ClO2 has recently come under

consideration as a possible root canal irrigant because of its similar reported

antibacterial activity in eliminating E.faecalis to NaOCl within 30 minutes on

dentinal discs. A study by Cobankara et al. in 2010 demonstrated that 13.8% ClO2

was as effective as 5.25% NaOCl in dissolving bovine pulp tissue.11

Although the combination of EDTA and Sodium Hypochlorite (NaOCl) have

been advocated as an effective irrigation regimen to remove the organic and the

Introduction

4

inorganic matter, a study by Deepa et al. in 2015 has shown that using 17% EDTA

solution also resulted in the maximum amount of calcium loss as the treatment time

increased, accompanied by a decrease in dentin microhardness.12 Since various

irrigants had to be used together to achieve the maximum effect, it resulted in dentists

spending a lot of time on the irrigation protocol alone. Hence, new irrigants were

introduced which has favourable properties as well as the same effect on the root

canal system, for better patient management and cleaning and shaping of the canal.10

BioPure MTAD marketed by Dentsply, Tulsa is a mixture of 3% tetracycline

isomer (doxycycline), 4.25% citric acid, and 0.5% detergent and has been shown to

be effective in the smear layer removal (Torabinejad et al., 2003).10 According to a

study by Kandil et al. in 2014, which evaluated the effect of different irrigating

solutions on the microhardness of root dentin, MTAD showed a lower effect on the

microhardness of root dentin than Malic acid and EDTA.13 Its action induced

inconsiderable erosion but promoted a significant decrease in dentin microhardness

(Dineshkumar et al., 2012; Saghiri et al., 2009).10 The results of the study differed

from other authors like Garcia Godoy et al. in 2005 who stated that both MTAD and

EDTA decrease the microhardness of root canal dentin because they collapsed the

dentin matrix structure. This effect by MTAD could be because of the presence of 3%

doxycycline hyclate component (an isomer of tetracycline) which acts as a calcium

chelator and causes root surface demineralization, as well as 4.25% citric acid which

dissolves mineral content of dentin.13

QMix 2in1 (Dentsply Tulsa Dental, Tulsa, USA) is a new irrigation solution of

antimicrobial agents for smear layer removal; it contains EDTA, CHX and a

Introduction

5

surfactant. The addition of a surfactant to chelating agents results in reduction of

dentin microhardness, but according to the study by G. Charul et al. in 2017, QMix

showed least reduction in microhardness of root canal dentin as compared to 17%

EDTA and MTAD, and has properties like biocompatibility, antimicrobial action, and

smear layer removal. Normally, the mixing of EDTA and CHX produces a white

precipitate, but in QMix 2in1 this is avoided because of its chemical design.10

The role of natural extracts for endodontic purpose in smear layer removal has

been evaluated for plants such as Morinda citrifolia and German chamomile

(Marticaria recutita) extract and tea tree (Melaleuca alternifolia) oil in a review of

Natural therapeutic option in Endodontics by V. Nagendrababu et al. in 2016.

Lahijani et al. in 2006 compared the chamomile hydroalcoholic extract and tea tree

oil to 2.5% NaOCl for smear layer removal. They concluded that chamomile has the

ability to remove the smear layer better when compared to NaOCl, but lesser than the

combination of NaOCl and EDTA. The smear layer removal could be attributed to the

presence of acidic components (capric acid, caprylic acid, chlorogenic acid, o-

caumaric acid, p-caumaricacid, dihydroxybenzoic acid) in the extract.14 The

antibacterial effects of German chamomile could be attributed to the presence of

active components like α-bisabolol and azulenes, which show anti-inflammatory

effects as well. More than 120 chemical constituents have been identified in the

flower as secondary metabolites, including 28 terpinoids, 36 flavonoids, and 52

additional compounds with potential pharmacological activity. Components like α-

bisabolol and cyclic ethers are antimicrobial, umbelliferone is fungistatic,

chamazulene is antiseptic, and flavonoids like apignine and luteoline are anti-

Introduction

6

inflammatory. Higher concentrations like 250mg/mL showed antimicrobial activity

against Enterococcus faecalis and Candida albicans too, but not more that 2%

CHX.15 To date, there are no studies comparing the effect of Marticaria recutita on the

root canal dentine as opposed to other root canal irrigants.

The structural properties of dentin, such as microhardness, permeability, and

solubility, may change after the use of chemical irrigants, which can alter the

proportion of organic and inorganic components. Microhardness is considered as

indirect evidence of mineral changes in root dentin; such changes could affect the

adhesive properties of dentin surface.16 In the specific case of root dentin, the agents

react with the calcium ions in the hydroxyapatite crystals. This process can cause

changes in the microstructure of the dentine and changes in the Ca:P ratio.17

Microhardness is sensitive to the composition and surface changes on the tooth

structure and Knoop hardness and Vicker’s Microhardness tester are responsible for

measuring the hardness of the dentin. Pashley et al. reported in a study conducted in

1985 that there is an inverse correlation between the dentin microhardness and tubular

density so, any significant alteration in the dentin microhardness after irrigation with

different chemicals will indicate the direct effect the chemicals have on the

components of the dentin structure.12

Previously studies have been conducted on Matricaria recutita (German

chamomile) to evaluate its effect on smear layer removal and on the disinfection of

the root canal space, but currently there are no studies that evaluate the effect of this

herbal product on the microhardness of root canal dentin.

Introduction

7

Thus, this study was aimed to evaluate for the first time, the effect Matricaria

recutita or German chamomile has on the microhardness of the root canal dentin and

to compare the effects with conventional irrigating solutions such as EDTA, ClO2,

and a novel irrigating solution QMix, which is already available in the market.

Objectives

8

The objectives of this in vitro study are:

i. To evaluate the effect of Matricaria recutita on the microhardness of the root

canal dentin.

ii. To compare and evaluate the difference in microhardness of root canal dentin

after exposure to the different root canal irrigants.

Null Hypothesis

The null hypothesis is that there is no significant difference between the effect of

EDTA, CHX, QMix, and Marticaria recutita when used as a final rinse, on the root

canal dentin.

Review of Literature

9

The objective of a successful endodontic treatment is to essentially remove all

vital or necrotic and gelatinous mass pulp remains that accumulate in the pulpal

chamber and the root canal wall in order to decontaminate it. This is achieved by

debridement with manual and mechanical instruments inside the canal along with

simultaneous irrigation. It is known that tissue debridement with manual or rotary

instruments produce a smear layer of about 2-2.5 mm thickness that accumulates on

the surface of the dentinal tubules and inside them and is composed mainly of

bacteria, toxins, hydroxyapatite crystals, saliva, and blood, making root canal

irrigation necessary for its success.7

With every thrust of an instrument, noxious materials like necrotic pulp or

shreds of mummified tissues are most likely to be pushed into the periapical area

through the apical foramen, resulting in possible periradicular pathologies. Thus

during each instrumentation it becomes important to irrigate simultaneously with

irrigating solutions capable of disinfecting them and dissolving organic and inorganic

material.18

Sodium hypochlorite is a salt formed by the bonding of chemical compounds

like hypochlorous acid (HOCl), and sodium hydroxide (NaOH). It is also hypertonic

and alkaline and has a pH higher than 11. NaOCl possesses certain good properties

like antibacterial action, dissolution of organic matrix, oxidisation and hydrolysis of

protein, and removal of intracellular fluids as well as magnesium and carbonate ions

present in root canal dentin. In addition, it also destroys fungi, spores, and viruses

when used at low concentration ranging from 1 to 5.25% without resulting in

toxicity.19 And although 5.25% sodium hypochlorite is used as a single irrigant in


10

most root canals, it does not have the capacity to completely remove the inorganic

portion of the dentinal smear layer and does not appropriately moisten the root canal

walls in narrow canals. Therefore, it has been proposed to alternate hypochlorite with

chelating agents that expand the root canal lumen facilitating access. The most widely

used is ethylenediaminetetraacetic acid (EDTA); and although this particular protocol

is the most effective for cleaning and shaping of root canals, it is still controversial on

the topic of reduction in microhardness of the root canal dentin.7

In its chemical structure, ethylenediaminetetraacetic acid, EDTA, has six

potential sites for binding metal ions and thus forms stable complexes with calcium

ions and demineralizes superficial root canal walls by simplifying the preparation of

narrow canals.20 However, it does not possess any disinfectant capacities and cannot

remove the organic component of dentinal smear. It is also believed that when

combined with sodium hypochlorite, it inactivates chlorine, removing its proteolytic

capacity.21

Goldberg and Abramovich in 1977 conducted a study analysing the effect of

EDTAC which reported that addition of a quaternary ammonium bromide (Cetavlon)

increased the action of EDTA by reducing its surface tension, because EDTA

solutions act only through direct contact with the substrate. This combination, known

as EDTA plus Cetavlon (EDTAC), was shown to be very effective in smear layer

removal and increasing the diameter of the opened dentinal tubules. Recently Çalt

and Serper in 2000 reported that ethylene glycol-bis [b-aminoethylether]-N,N,N=,

N=-tetraacetic acid (EGTA) was also effective in removing the smear layer, without

inducing dentinal erosion commonly caused by EDTA. Tetracycline-hydrochloride


11

(HCl) has also been proposed as a root canal irrigant and in addition to its

antimicrobial effect tetracycline-HCl also acts as a calcium chelating agent due to its

low pH. One percent tetracycline HCl has been shown to be as effective as 50% citric

acid in the removal of smear layer, while causing less demineralization of peritubular

dentin.1

Dogan et al. in 2001 reported that some chemicals used for endodontic

irrigation are capable of causing alterations in the chemical composition of dentin and

that any change in the Ca:P ratio may alter the original proportion of organic and

inorganic components, which in turn could change the microhardness, permeability,

and solubility characteristics of dentin.22 Studies have also shown that different

concentrations of EDTA, EDTAC, and EGTA are capable of decreasing the

microhardness of root canal dentin and that this effect can increase by extended

application times. Changes in the mineral content of superficial dentin may also

adversely affect the sealing ability and adhesion of dental materials such as resin-

based cements and root canal sealers to dentin.23

Effect of EDTA on microhardness

A study aimed to evaluate the effect of citric acid, ethylenediaminetetraacetic

acid (EDTA) and ethylenediaminetetraacetic acid plus Cetavlon (EDTAC) solutions

on the microhardness of human root canal dentine. Sixteen maxillary human canines

were sectioned transversely at the cemento-enamel junction and the crowns were

discarded. Subsequently, each root was embedded in an epoxy resin cylinder and their

middle third sectioned horizontally into 4 mm thick slices. The samples were


12

randomly divided into three groups according to the chelating agent employed, as

follows (n = 6): group 1: EDTA 17%, group 2: EDTAC 17% and group 3: citric acid

10%. Dentine microhardness was then measured with a load of 50 g for 15 s. At the

beginning of the experiment, reference microhardness values were obtained for

samples without any etching (t = 0 min). The same samples were then exposed to 50

microL of the chelator solution for 1, 3 and 5 min. The Student's t-test (P < 0.05) was

used to compare results for different times for each chelator and different chelator for

each time. Microhardness decreased with increasing time of application of chelating

solutions. There were no significant (P > 0.05) differences between initial

microhardness for the three groups as well as after 1 min of application of the

substances. After 3 min, EDTA produced a significantly greater reduction in

microhardness. However, there was no significant difference between EDTA and

EDTAC after 5 min. Citric acid caused significantly less reduction in microhardness.

Overall, citric acid was least effective in reducing dentine hardness whilst EDTA had

the strongest effect.17

A study evaluated the effect of single and combined use of ethylenediamine

tetra acetic acid (EDTA), ethylene glycol bis [b-aminoethylether] N,N,N=,N=-

tetraacetic acid (EGTA), EDTA plus Cetavlon (EDTAC), tetracycline-HCl, and

NaOCl on the microhardness of root canal dentin. The crowns of 30 single-rooted

human teeth were discarded at the cementoenamel junction and the roots were

bisected longitudinally to obtain root halves (N 60). The specimens were embedded

in auto polymerizing acrylic resin, leaving the root canal dentin exposed. Dentin

surfaces were prepared for microhardness test by grinding and polishing. The


13

reference microhardness values of untreated specimens were recorded using a

Vicker’s microhardness tester at the apical, mid-root, and cervical levels of the root

canal. Thereafter, the specimens treated with single (test solution only) or combined

(test solution, followed by 2.5% NaOCl) versions of the irrigants for 5 minutes. Post

treatment microhardness values were obtained as with initial ones. Statistical

comparisons between the test groups and among single and combined treatments

were carried out using 2-way ANOVA with repeated measures (P .05). Comparisons

within each group with respect to application regions were made with Friedman’s

nonparametric 2-way analysis of variance at the same level of significance. It was

found that, all treatment regimens except distilled water significantly decreased the

microhardness of the root canal dentin (P. 05). The single and combined use of EDTA

decreased the microhardness of the root canal dentin significantly more than all other

treatment regimens (P .05). Compared with their single-treatment versions, all

combined treatment regimens decreased the mean microhardness values significantly

(P .05). A comparison of single and combined treatment regimens revealed

significant decreases only for EDTA and EDTA NaOCl in the coronal region and for

EDTAC and EDTAC NaOCl in the apical and middle regions of the root canal (P

.05). Therefore they concluded that the use of EDTA alone or prior to NaOCl resulted

in the maximum decrease in dentin microhardness. The softening effect of subsequent

NaOCl treatment was both material and region dependent. However, for combined

treatment regimens, subsequent use of NaOCl levels the statistical differences

between the regional microhardness values obtained after treatment with EGTA,

EDTAC, and tetracycline-HCl.1


14

The aim of an in vitro study was to evaluate the effect of 7% maleic acid and

17% EDTA solutions on the microhardness and the surface roughness of human root

canal dentin. Forty-five extracted human maxillary central incisors were sectioned

longitudinally into a total of 90 segments, which were embedded in auto

polymerizing acrylic resin, and were grounded flat with silicon carbide abrasive

papers. Based on the test solutions used, samples were divided randomly into three

groups: (1) the EDTA group, 1 mL of 17% EDTA for 1 minute (n = 30), (2) the

maleic acid group, 1 mL of 7% maleic acid for 1 minute (n = 30), and (3) the control

group, 1 mL of 0.9% saline for 1 minute (n = 30). Every group was then divided into

two subgroups of 15 specimens each. In group 1a, 2a, and 3a, specimens were used to

determine the microhardness of the root canal dentine in the coronal, middle, and

apical third using Vicker's hardness tester. In groups 1b, 2b, and 3b, specimens were

used for the determination of surface roughness of the root canal dentine using a

roughness tester (Surtronic, Leicester, England). The data were statistically analyzed

using the Kruskall Wallis and Mann Whitney U tests. There was no significant

difference between EDTA and maleic acid in the reduction of microhardness. The

increase in roughness was significantly greater with maleic acid when compared with

EDTA. Maleic acid reduced the microhardness of root dentin similar to EDTA but

increased the surface roughness significantly more than EDTA.24

Another study aimed to evaluate the microhardness of human dentin by using

four irrigating solutions. A total of 40 extracted mandibular premolars were selected

and sectioned horizontally in the middle third of the root. Forty specimens of 4 mm

thickness were embedded in acrylic resin and polished. Four test groups, each group


15

containing ten specimens were immersed in respective irrigating solution and

subjected to Vicker’s microhardness test at T0, T2 and T5min. The data obtained

were analyzed using the one way ANOVA followed by Tukey HSD method with

p=0.05 as the level for statistical significance. The results suggested that there was no

statistically significant difference in mean values between four experimental

irrigating solutions. Mixture of Tetracycline isomer i.e. Doxycycline, Citric acid and

a Detergent (Tween 80) MTAD did not alter the microhardness of root canal dentin

significantly and seemed to be an appropriate irrigating solution, because of its

harmless effect on the microhardness of the root canal dentin.2

An in vitro study was conducted to compare the effect of different irrigants on

root dentin microhardness and smear layer removal. A total of 50 roots were equally

divided into two halves to measure dentin microhardness and to evaluate the amount

of smear layer. One hundred root halves were divided into five equal groups 20

sample each according to the final irrigants used: Group 1: 2.5% NaOCl, Group 2:

2.5% sodium hypochlorite (NaOCl) followed by 7% malic acid (MA), Group 3: 2.5%

NaOCl followed by 17% ethylenediaminetetraacetic acid (EDTA), Group 4: 2.5%

NaOCl followed by mixture of tetracycline, acid and detergent (MTAD) and Group 5:

saline. Ten root halves from each group were prepared to measure dentin

microhardness at baseline measurement and after treatment to determine the change

in microhardness, while the remains 10 root halves were prepared for

scanning electron microscope to evaluate the amount of smear in the coronal, middle

and apical thirds. Data were analyzed using one-way ANOVA and Student's t-test for

microhardness and Kruskall–Wallis and Mann–Whitney for smear layer. Malic acid

https://www.sciencedirect.com/topics/medicine-and-dentistry/ethylenediamine

https://www.sciencedirect.com/topics/medicine-and-dentistry/tetracycline

https://www.sciencedirect.com/topics/medicine-and-dentistry/electron-microscope


16

showed the greatest significant reduction in dentin microhardness (P < 0.05),

followed by EDTA, MTAD, NaOCl and saline (control). EDTA, malic acid and

MTAD efficiently removed smear layer, respectively, in the coronal and middle

thirds of root canal. However, in the apical region, malic acid showed more efficient

removal of the smear layer than the other irrigants. As a conclusion, Malic acid is the

most efficient final irrigant solution after NaOCl irrigation throughout

instrumentation.13

A study aimed to evaluate the effects of root canal irrigants on the

microhardness of root canal dentin in the presence and absence of surface-modifying

agents. Forty-eight root halves were prepared by longitudinal splitting of the distal

roots of 24 freshly extracted mandibular human third molars and embedded in auto

polymerizing acrylic resin, leaving the dentin surface exposed. After polishing, the

microhardness values of the untreated dentin surfaces were recorded by using Vickers

tester at the mid-root level. The root halves were randomly assigned to 6 groups

composed of 8 samples each and treated for 5 minutes with one of the following

irrigants: 17% EDTA, REDTA, 2% chlorhexidine gluconate (CHX), 2% CHX with

surface modifiers (CHX-Plus), 6% NaOCl, or 6% NaOCl with surface modifiers

(Chlor-XTRA). After surface treatment, dentin microhardness values were recorded

at close proximity to the initial indentation areas. Experimental data were statistically

analyzed by using the t test and one-way analysis of variance, followed by Tukey

honestly significant difference test at α = 0.05. EDTA, REDTA, NaOCl, and Chlor-

XTRA significantly decreased the microhardness of root dentin compared with intact

https://www.sciencedirect.com/topics/medicine-and-dentistry/root-canal


17

controls (P < .05). The addition of surface modifiers to the irrigants did not affect the

microhardness of the samples.25

A study aimed to evaluate the influence of the following irrigating solutions on

the microhardness of root canal dentin: 2% sodium hypochlorite (2NaOCl), 5%

sodium hypochlorite (5NaOCl), super-oxidized water (400 ppm Sterilox - Sx) and

17% EDTA (E). Eighty roots from bovine incisors were randomly divided into 8

groups (n=10): 2NaOCl, 5NaOCl, Sx, and 2NaOCl + E, 5NaOCl + E, Sx + E

(associated with E as final irrigant for 5 min), E solely and distilled water (dH2O) as

the negative control. Root canal preparation was performed by hand instruments,

using one of the irrigation protocols for 30 min. Then, 5 mm of the cervical root third

were cut out from each sample and subjected to the Vickers microhardness test, at

two points, one at approximately 500-1000 µm from the root canal lumen (distance

1), and the other at approximately 500-1000 µm from the external root surface

(distance 2). Data were analyzed by Wilcoxon and Kruskal-Wallis tests at 5%

significance level. Microhardness values at distance 1 were significantly lower than

those at distance 2 for all groups, except 5NaOCl and 5NaOCl + E groups (p>0.05).

EDTA showed the lowest microhardness values. However, no statistically significant

difference was detected among groups at distance 1 and EDTA was significantly

different only from Sx at distance 2. In conclusion, all tested solutions showed lower

microhardness at the most superficial root canal dentin layer compared to the one

found near the external root surface, except 5NaOCl and 5NaOCl + E; EDTA

promoted lower microhardness values in comparison to Sterilox at this site.26


18

Another study was done to determine the Calcium loss and its effect on

microhardness of the root canal dentin following treatment with aqueous solution of

17% Ethylenediaminetetraacetic acid at different time intervals. In this study, twenty

extracted human canine teeth were bisected longitudinally and root halves were

embedded individually in rings with polyester resin. Samples were randomly divided

into 7 groups based on different time intervals i.e., each group had 5 samples each

(n=5 samples) and the remaining 5 samples served as a negative control group.

Initially, 20 ml of 17% EDTA solution was prepared as a blank to determine the

calcium levels in absence of the specimen. Then each sample was immersed in a

beaker containing 20 ml of experimental solution according to the different time

intervals and maintained under constant agitation using a magnetic multi-stirrer

(SBSA-09 series C, Barcelona, Spain) to homogenize the extracted calcium in the

solution. Then the samples were taken out of the beaker after the respective time

interval. Five ml aliquots were extracted from the beaker using a calibrated micro

pipette. These extracts were then placed in hermetically sealed and labeled glass

tubes. By this way, extracts were obtained for each sample at different time intervals.

The same samples were subjected for the evaluation of amount of Ca2+ release into

the solution by Atomic Absorption Spectrophotometer. Then the microhardness of the

sample used for determining the Ca2+ loss was evaluated at different dentin surface

adjacent to the root canal lumen in a microhardness testing machine (Micromet 2100

Microhardness Tester, Buehler). All experiments were completed under the same

conditions: 50 g load and 15s dwell time, following the guidelines given by Cruz-

Filho et al. The results were statistically analyzed (p<0.05) showing the time


19

dependant effect of 17% EDTA in both calcium loss as well as microhardness

reduction in comparison with saline (control group). And thus it was concluded that,

root canal irrigation with 17% EDTA solution is time dependant, increased irrigation

time leads to structural changes, as evidenced by reduction of dentin microhardness.12

An in vitro study was carried out to compare of the effect of 17%

ethylenediamine tetra-acetic acid (EDTA), 2% chlorhexidine (CHX), 18% etidronic

acid (HEBP), and 4% propolis as an irrigant on the microhardness of root dentin. The

sample size for the study was 100. Each specimen consisted of a longitudinally

sectioned half of a root of a single-rooted tooth which was embedded in acrylic resin.

The prepared specimens were divided randomly into five groups of twenty specimens

each. Each group was treated with the irrigants to be tested. Group I was the control -

the specimens were treated with distilled water. The specimens in Group II were

treated with sodium hypochlorite (NaOCl) followed by EDTA. Specimens in Group

III were treated with NaOCl followed by CHX. Specimens in Group IV were treated

with NaOCl followed by HEBP, and specimens in Group V were treated with NaOCl

followed by propolis. Following this, all the specimens were placed on the Vickers

hardness tester and three readings were taken for each specimen. An average reading

was obtained for each group. The results were tabulated and statistically analyzed to

determine which of the irrigant solutions had the least effect on the microhardness of

root dentin. Eighteen percent HEBP had the least effect on the root dentin

microhardness, followed by 4% propolis and 2% CHX. Seventeen percent EDTA

showed maximum effect on the microhardness of the dentin. Under the limitations of


20

this study, 18% HEBP and 4% propolis show promise for use as irrigants because of

less detrimental effect on the hardness of root dentin.27

An in vitro study evaluated the effects of root canal irrigants on the

microhardness of root canal irrigants on the microhardness of root canal by using

three types of irrigant solutions with different concentration and normal saline used as

control group. In this study the root halves were prepared by longitudinal splitting of

the roots of 56 freshly extracted caries free maxillary second premolars and

embedded in auto polymerizing acrylic resin, leaving the dentin surface exposed. The

root halves were randomly divided to seven groups composed of 16 samples each and

treated for 5 min with one of the following irrigants: normal saline (control group),

0.2% chlorhexidine, 2% chlorhexidine, 2.5% sodium hypochlorite, 5.25%, 5%

ethylene dimetha tera hydrate EDTA and 17% EDTA. After surface treatment, the

dentin microhardness of the root samples was recorded at the mid-root level by using

vicker’s microhardness tester. The data were statistically analyzed by using one-way

analysis of variance, followed by Duncans test with a significant difference test at

p<0.05. The results showed that EDTA, sodium hypochlorite, and 2% chlorhexidine

significantly decreased the microhardness of root dentin compared with controls

(p,0.05), while 0.2% chlorhexidine had no significant effect on the microhardness of

root dentin. Therefore, it was concluded that the irrigant solutions affect the

microhardness of the samples except 0.2%.28

Another study was conducted to study the microhardness of root canal dentin

after irrigation with different irrigant solutions for different periods. Twenty five

newly extracted non carious human permanent incisors were sectioned at


21

cementoenamel junction and split longitudinally then divided into five groups; Gr1

(control) distilled water, G2: 5.25% sodium hypochlorite (NaOCl) for (10 min) then

17% EDTA for (1 min), G3: 5.25% sodium hypochlorite (NaOCl) for (10 min) then

17% EDTA for (5 min), G4: 5.25% sodium hypochlorite(NaOCl) for (20 min) then

17% EDTA for (1 min) and G5: 5.25% sodium hypochlorite(NaOCl) for (20 min)

then 17% EDTA for (5 min). Vickers microhardness was evaluated. Data were

analyzed using one-way ANOVA and paired t-test. The results indicated that all

treatment time with 5.25% NaOCl and 17% EDTA decreased dentin microhardness

significantly compared to distilled water (control). There were significant differences

(P<0.001) between the tested groups with increasing time of exposure of irrigation

solutions. Treatment with distilled water (control) showed significantly the highest

microhardness value, while 5.25% sodium hypochlorite for 20 minute followed by 5

minutes (G5) with 17% EDTA showed significantly the least microhardness value

followed by G4, G3 and G2. Therefore it was concluded that, increasing irrigation

time with both 5.25% sodium hypochlorite and 17% EDTA decreased dentin

microhardness.6

A study was conducted to evaluate the effect of different irrigation protocols on

microhardness of human root canal dentin. Forty extracted single rooted lower

premolars were used. All teeth were instrumented using manual stainless steel files

and irrigated by 2ml distilled water between each file, then were sectioned by

longitudinal splitting of each tooth. The root halves were randomly assigned into 4

parallel groups (n=10) and immersed for 5 minutes with one of the following

irrigants: Group I: 10 ml of 2.5% Sodium Hypochlorite (NaOCl), Group II: 10 ml of


22

17% ethylene diamine tetra-acetic acid (EDTA) followed by 10 ml of 2.5% NaOCl,

Group III: 10 ml of 2.5% NaOCl followed by 10 ml of 2% chlorhexidine di gluconate

(CHX), Group IV: 10 ml of 2.5% NaOCl followed by 10 ml distilled water then were

followed by 10ml of 2% CHX. Ten root halves from each group were prepared to

measure dentin microhardness at baseline measurement and after treatment to

determine the change in microhardness, using Vickers tester. Data were analysed

using t-test, ANOVA test and Post Hoc test. Group II showed the highest percentage

decrease in microhardness values, followed by group III, then group IV and the

lowest was group I. All groups showed a significant difference between each other (P

< 0.05), except group III and IV. The coronal third showed the highest percentage

decrease with significant difference between apical and middle thirds (P < 0.05), in

which there was no significant difference between them. In conclusion, CHX is the

best final irrigant if there is excellent intermediate flush for prevention of its

precipitation with NaOCl. The coronal third needs conservative approach as it is the

most affected third.28

The aim of a study was to evaluate the effect of various endodontic irrigants on

the micro-hardness of the root canal dentin. This in vitro study was carried out on

eighty freshly extracted mandibular premolars with single canals. They were

decoronated at the cemento-enamel junction. Roots were sectioned longitudinally into

two halves. They were then polished and placed in auto polymerized resin moulds

with the polished surface facing outside. The samples were divided into four groups

based on the irrigants in which they were immersed i.e., 3% Sodium Hypochlorite

(3% NaOCl), 17% Ethylene Dioxide Tetra Acetic Acid (17% EDTA), 0.2% Chitosan


23

and 6% Morinda citrifolia Juice (MCJ) for 15 minutes each. All the specimens were

then subjected to micro-hardness testing using a Vickers micro-hardness tester.

Statistical analysis was done using one way Analysis of Variance (ANOVA), Post-

Hoc Tukey test and Paired t-test to compare the pre and post immersion micro-

hardness values of the selected samples. The results of the present study indicated that

17% EDTA and 0.2% Chitosan significantly decreased the micro-hardness of root

dentin whereas 6% MCJ and 3% NaOCl had no significant effect on the

microhardness before and after immersing in the irrigants. A 6% MCJ and 3% NaOCl

which have significant antibacterial, antifungal, anti-inflammatory and smear layer

removing properties showed negligible effect on the micro-hardness of root canal

dentin making them suitable endodontic irrigating solution.

Effect of Chlorine Dioxide on microhardness

A study evaluated the effect of chlorine dioxide and various other more

common irrigation solutions on the microhardness and surface roughness of root

canal dentin. Fifty human maxillary central incisors were sectioned longitudinally and

treated for 1 minute with 5 ml of the following aqueous solutions (v/v%): Group 1:

13.8% chlorine dioxide, Group 2: 17% ethylene diamine tetraacetic acid (EDTA).

Group 3: 7% maleic acid, Group 4: 2.5% sodium hypochlorite (5ml/min), Group 5:

Saline (control). Specimens were subjected to microhardness and surface roughness

testing. Chlorine dioxide and sodium hypochlorite reduced the microhardness more

than other test agents. The highest surface roughness was produced with maleic acid.

Chlorine dioxide should be used cautiously during chemo-mechanical preparation of

the root canal system in order to prevent untoward damage to the teeth.11


24

Effect of QMix on microhardness

A study aimed to compare the changes in microhardness of root dentin caused

by two novel irrigation regimens with conventional irrigation. Forty extracted human

permanent incisor teeth were selected. Decoronated roots were separated

longitudinally to get 80 specimens that were embedded in auto polymerizing acrylic

resin and grounded flat with silicon carbide abrasive papers. Of these, 60 root

segments without any cracks or defects were selected and divided into four groups

according to the irrigation regimen used (n = 15). Group I: 5% sodium hypochlorite

(NaOCl) + 17% ethylenediaminetetraacetic acid (EDTA) + 0.2% chlorhexidine di

gluconate (CHX) (conventional). Group II: 6% Morinda Citrifolia Juice + 17%

EDTA (MCJ). Group III: 5% NaOCl + Q Mix 2 in 1 (QMix). Group IV: Distilled

water (control). Irrigation regimens were performed for 5 minutes. Dentin

microhardness was measured with a Vickers indenter under a 200-g load and a 20-s

dwell time at the mid-root level of root dentin. The data were analyzed using Kruskal

Wallis test and Dunn's multiple comparison tests. A significant difference was seen in

the median values of the four groups. The control group showed the least reduction in

microhardness when comparison with the other groups. Except for Group III (Q Mix),

the other groups that were tested (MCJ and conventional regimens) showed

statistically significant difference from the control group. In conclusion, within the

limitation of this study, it was concluded that NaOCl + Q Mix were least detrimental

to root dentin microhardness when compared with MCJ and conventional irrigation

regimens.30


25

Another study evaluated the effects of QMix, EDTA + CHX, EDTA + NaOCl

and maleic acid on the microhardness of root canal dentine. Forty recently extracted

human maxillary canine teeth were longitudinally sectioned into 80 segments and

then embedded in an auto polymerizing acrylic resin. The microhardness of the

dentine in the specimen was measured with a Vickers diamond indenter at the

Coronal, middle and apical thirds of the roots. Finally, the specimens were divided

randomly into four groups: 17% EDTA + 2.5% NaOCl; 17% EDTA + 2% CHX;

QMix; and 7% maleic acid. Post-treatment microhardness values were obtained and

the decrease in microhardness was calculated as a percentage. Microhardness values

were statistically analysed using the Kruskal-Wallis and Mann-Whitney U tests.

According to this study Maleic acid significantly decreased microhardness in all

regions, compared to the other groups. In the coronal and middle regions, there was

no significant difference among the other groups. In the apical region, there was no

significant difference between QMix and 17% EDTA + 2% CHX but these groups

presented significant dentine microhardness reduction compared to the 17% EDTA +

2.5% NaOCl group. In conclusion, while maleic acid showed the greatest reduction in

dentine microhardness, it was found that QMix, 17% EDTA + 2% CHX and 17%

EDTA + 2.5% NaOCl cause the same reduction in the microhardness of root canal

dentine in the coronal and middle regions.31

Another study aimed to evaluate the effect of final irrigation protocols (17%

EDTA, BioPure MTAD, SmearClear, and QMix) on microhardness and erosion of

root canal dentin. Fifty roots were sectioned transversely at the cemento-enamel

junction and each root was sectioned horizontally into 4-mm-thick slices. The


26

samples were divided into five groups (n = 10) according to the final irrigation

protocol: G1: distilled water (control group); G2: 17% EDTA; G3: BioPure MTAD;

G4: SmearClear; and G5: QMix. The dentin microhardness was then measured with a

load of 25 g for 10 s. Initially, the reference microhardness values were obtained for

the samples without any etching. The same samples were then submitted to the final

irrigation protocols. A new measure was realized and the difference between before

and after the procedures was the dentine microhardness reduction. In sequence, the

specimens were submitted to SEM analysis to verify the dentinal erosion. The

Kruskal Wallis and Dunn tests (α = 5%) were used to compare the results. The dentin

microhardness decreased for all final irrigation protocols. There was no significant

difference between groups 2, 3, 4, and 5 (P > 0.05), but these groups presented

significant dentine microhardness reduction than G1 (P < 0.05). In G2, occurred the

highest incidence of dentinal erosion (P < 0.05). 17% EDTA, BioPure MTAD,

SmearClear, and QMix promoted significant dentine microhardness reduction.

Dentinal tubules erosion was promoted by 17% EDTA.32

A study aimed to evaluate the effect of final irrigation protocols on

microhardness reduction and erosion of root canal dentin. Sixty root canals from

mandibular incisors were instrumented and randomly divided into six groups (n = 10)

according to the irrigant used: QMix, 17% EDTA, 10% citric acid (CA), 1% peracetic

acid (PA), 2.5% NaOCl (solution control), and distilled water (negative control). The

chelating solutions were used to irrigate the canal followed by 2.5% NaOCl as a final

flush. After the irrigation protocols were completed, all the specimens were rinsed

with 10 mL of distilled water to remove any residue of the chemical solutions. Before


27

and after the final irrigation protocols, dentin microhardness was measured with a

Knoop indenter. Afterwards, the specimens were prepared for scanning electron

microscopic analysis and the amount of dentin erosion was examined. Wilcoxon and

Kruskal-Wallis tests were used to analyze the results with a significance level set at

5%. At 100 µm, all protocols significantly reduced dentin microhardness (p < .05),

while at 500 µm, this effect was detected only in the EDTA and QMix groups (p <

.05). CA was the irrigant that caused more extensive erosion in dentinal tubules,

followed by PA and EDTA. QMix opened dentinal tubules, but did not cause dentin

erosion. Results suggest that QMix and 17% EDTA reduced dentin microhardness at

a greater depth. Additionally, QMix did not cause dentin erosion.33

A study aimed to evaluate and compare the effect of QMix, Tea tree oil,

Tamarindus indica, Green tea extract and 17% EDTA on root dentine micro-hardness.

Sixty freshly extracted human single rooted premolars were selected and divided into

six groups and subjected to various treatments as follows: Group 1-Q mix, Group 2-

Tea tree oil, Group 3 - 5% Tamarindus indica, Group 4 - 5% Green tea, Group 5 -

17% EDTA and Control group-normal saline. Each group was immersed in their

solutions for 5 minutes and then subjected to Vickers micro-hardness testing.

According to this study, maximum reduction in micro-hardness was seen in the

EDTA group, followed by QMix and then Tamarindus indica groups. Tea tree oil

group and Green tea groups did not show significant reduction in microhardness.

Least reduction was seen in the control group saline. In conclusion, EDTA induced

maximum reduction in root dentin microhardness, followed by QMix and Tamarindus


28

indica. There was no significant reduction by Green tea, Tea tree oil and saline (p >

0.05) Tamarindus indica caused significantly less reduction than EDTA (p < 0.001).34

Vicker’s Microhardness testing on root dentin

In another study, Vickers micro hardness was assessed on root dentin and

cementum in 30 canines obtained from the Periodontics Discipline tooth bank. Crown

and cervical portion of the root were discarded by a transverse section situated 3mm,

apically, from the cemento-enamel junction on the buccal surface, thus obtaining the

root specimen. On root dentin, mean micro hardness values were obtained for 3

regions: (DR1) from 0.05 to 0.1 mm of the cemento dentin junction; (DR2) from 0.3

to 0.5 mm of the cemento dentin junction and (DR3) from 0.8 to 1.0 mm of the

cemento dentin junction. After statistical analysis (Variance Analysis test, p < 0,05),

we conclude that micro hardness at the DR2 region was statistically greater than that

at the DR3 region which was, in turn, greater than that at the DR1 region. The mean

micro hardness of cementum was 19.70 VHN.35

Another study aimed to determine the effect of variations in indentation load

and time on the Knoop and Vickers hardness numbers (KHN and VHN) for enamel

and dentin. Twenty molar teeth were divided into twenty enamel and twenty dentin

specimens. Each specimen was tested using a Knoop or Vickers microhardness tester

at different loads and times. The difference in hardness between the groups was

analyzed with two-way ANOVA followed by a Tukey test. The results revealed that a

difference of indentation time did not influence the microhardness number of enamel

and dentin. The KHN values of enamel and the VHN values of dentin were affected


29

by variation of test loads. Therefore, the tooth hardness number for different loads

may not be acceptable for comparison.36

A study aimed to measure the microhardness of root dentin after

instrumentation with different file types and using irrigation with 2.5% sodium

hypochlorite. Root canal of 10 roots were irrigated only, root canal of 10 roots were

instrumented with stainless steel files, root canal of 10 roots were instrumented with

rotary nickel titanium files. Root canals of 10 roots were instrumented with nickel

titanium K-files. Additionally, five roots pulp was extirpated only, without irrigation

and served as control. The roots were sliced and root dentin microhardness was

determined at distance of 500 mm and 1000 mm from the pulp dentin interface. There

was a significant difference found between the microhardness at 500 mm and 1000

mm (P<0.001) in all groups. Instrumentation with Nickel titanium rotary files

affected dentin microhardness significantly to a lesser extent when compared to

stainless steel K files and Nickel titanium K-File. Therefore it was concluded that

instrumentation and irrigation with NaOCl changes the biomechanical properties of

dentin.3

Materials and Methodology

30

Source of Data

This study was conducted in the Department of Conservative Dentistry and

Endodontics, The Oxford Dental College, Bangalore.

Forty single rooted premolar teeth were collected from the Department of Oral

and Maxillofacial Surgery, The Oxford Dental College, Bangalore. Ethical clearance

was obtained from the institutional ethical committee of The Oxford Dental College,

Bangalore. All patients were explained about the need for their extracted teeth and

informed consent was taken for dental research purpose.

Inclusion criteria

Single rooted human premolar teeth

Presence of single canal

Exclusion criteria

Direct and indirect restorations

Cracks

Fractured

Grossly decayed

Resorption

Dilacerations


31

Method of collection of data

The sample size was calculated based on the following criteria,

ANALYSIS: A priori: Compute required sample size

INPUT: Effect size f = 0.40

α err prob = 0.05

Power (1-β err prob) = 0.80

Number of groups = 5

OUTPUT: Noncentrality parameter λ = 12.8000000

Critical F = 2.4936960

Numerator df = 4

Denominator df = 75

Total Sample size = 80

Actual power = 0.8030845

The sample size has been estimated using the software Gpower v. 3.1.9.2.

Considering the effect size to be measured (d) at 40%, power of the study at 80% and

the margin of error at 5%, the total sample size needed is 80. So, each study group

will comprise of 16 samples.


32

Study protocol followed

Forty single rooted premolar teeth were selected and radiographs were taken to verify the

presence of a single canal.

To ensure standardization, the crowns of the teeth were removed at the cemento-enamel

junction, for a length of 18 mm.

Access cavities were prepared using diamond burs with a high speed hand piece under

water cooling and working length was determined with size #10K file

40 roots were instrumented with the Pro Taper Universal up to size 30 (6%) and irrigation

was done with distilled water between each instrument change.

The teeth were then split longitudinally into two separate buccal and lingual halves and

embedded in resin blocks with the dentin exposed.

The teeth were then randomly divided into 5 groups, one control group and 4

experimental groups.

Group I

EDTA (n=16)

Group II

ClO2 (n=16)

Group III

QMix (n=16)

Group IV

M.recutita(n=16)

Group V

Saline (n=16)


33

Instruments / Equipments (Table 1)

Equipment Purpose of use Manufacturer

Airotor hand

piece

Tooth

preparation

NSK, Inc Japan

Endomotor Root canal

preparation

Dentsply

Sirona, India

Vicker’s

Microhardness

Tester

Microhardness Struers, Gatan,

USA

Isomet Tooth

sectioning

Buehler, Lake

Bluff, IL, USA

The teeth were then treated with 3% NaOCl for 5 min, as an initial rinse and for 5 min

with each irrigant, as a final rinse.

The teeth were then subjected to a 100g load for a dwell time of 15 sec using Vicker’s

Microhardness Tester.

The two diagonals of the indentation left in the surface of the material after removing the

load were measured under microscope and the average calculated.

The area of the sloping surfaces of the indentation was calculated. Vicker’s hardness is

the quotient obtained by dividing the kgf load by the square mm area of indentation.


34

Materials (Table 2)

Material Purpose of use Manufacturer Batch No.

Single rooted

teeth

Specimen

Burs Access

preparation

Mani. Inc

Japan

K-files Biomechanical

preparation

Mani. Inc

Japan

Pro Taper

Universal files

Biomechanical

preparation

Dentsply

Maillefer, USA

Acrylic resin Embedding tooth

3% Sodium

hypochlorite

Irrigation 23286

Saline (0.9%

N.W.)

Irrigation IA80092

Paper Points Drying the canal Dentsply

Maillefer, USA

17% EDTA Irrigation Pulpdent 17042602

0.2% Chlorine

Dioxide

Irrigation Solumium

Dental,

Hungary

H270514

QMix Irrigation Dentsply, Tulsa 170519

Matricaria

recutita

Irrigation Biocarve, India


35

Composition of irrigants (Table 3)

EDTA E.D.T.A. di-sodium salt, distilled water

QMix Chlorhexidine and EDTA

Root canal Treatment

The teeth were stored in Hydrogen peroxide for a week and then in normal

saline until further use. They were thoroughly rinsed prior to use and the residual pulp

tissue was removed using barbed broaches. The working length for each tooth was

determined by inserting International Standard Organization (ISO) 10 K-file into the

canal and each canal was prepared till the working length with the Pro-Taper

Universal rotary system up to size 30 (6%) taper. The canals were then irrigated with

distilled water between each instrument change to avoid any unwanted changes to the

root canal dentin before conducting the study.

To ensure standardization of 18 mm, the teeth were decoronated at the

cemento-enamel junction using low speed diamond points and under copious water

irrigation to prevent overheating. The teeth were then sectioned longitudinally into

two halves and the separated buccal and lingual halves were then embedded in acrylic

resin, leaving the dentin surface exposed. The dentin surface was then polished using

silicon carbide abrasives to remove any surface irregularities, under distilled water

(prevent overheating).

The teeth were then randomly assigned to 5 groups, one control group and 4

experimental groups.


36

Preparation of Matricaria recutita

10g of the weighed plant material were soaked in 100 mL of hot water and

boiled for 30 min in a conical flask for 24 hrs. The solution was then filtered using

filter paper.

Sample treatment

All the samples were initially treated for 5 min with 3% NaOCl as an initial

rinse for its bactericidal properties. The specimens were then randomly divided into 5

groups (n=16) and treated for 5 min with 5 mL of one of the following irrigants:

Group I was cleaned and shaped using the protocol mentioned above and treated

with 17% EDTA (Prime Dental Products, India)

Group II was cleaned and shaped using the protocol mentioned above and treated

with 0.2% Chlorine Dioxide (Solumium Dental, Hungary)

Group III was cleaned and shaped using the protocol mentioned above and treated

with QMix (Dentsply, Tulsa)

Group IV was cleaned and shaped using the protocol mentioned above and treated

with Matricaria recutita (Biocarve, India)

Group V was cleaned and shaped using the protocol mentioned above and treated

with Saline (Control group)

After the surface treatment, the samples were washed and dried, and the dentin

microhardness of each sample was determined as described below.


37

Determination of dentin microhardness

The dentin microhardness of the root samples was initially measured using

Vickers microhardness tester. The sections were subjected to a 100g load for a dwell

time of 15s. In each sample, three indentations were made along the lines parallel to

the edge of the root canal lumen. The two diagonals of the indentation left in the

surface of the material after removal of the load was measured and their average

calculated. The area of the sloping surfaces of the indentation was calculated and the

Vickers microhardness is the quotient obtained by dividing the kgf load by the square

mm area of indentation.

F- Load in kgf

D- Arithmetic mean of the two diagonals, d1 and d2 in mm

HV- Vickers hardness

HV= 2Fsin 136°/2 HV= 1.854 F/d2

d2


38

Statistical Analysis

Statistical Package for Social Sciences [SPSS] for Windows Version 22.0 Released

2013. Armonk, NY: IBM Corp. was used to perform statistical analysis.

Descriptive Statistics:

Descriptive analysis includes expression of Micro Hardness in terms of Mean & SD.

Inferential Statistics:

One-way ANOVA test followed by Tukey's HSD post hoc Analysis was used to

compare the mean Micro Hardness values between study groups.

The level of significance was set at P<0.05.

Results

41

The Vicker’s microhardness number (VHN) values of all the tested groups after

treatment with the different irrigating solutions have been represented in individual

tables in the annexure. All the irrigating solutions decreased the microhardness of the

canal dentin surface, with EDTA showing the highest degree of reduction. The mean

diagonal of the indentation was determined in each of the sixteen samples of all the

five groups, and the mean microhardness values were calculated using the formula.

(Table 1)

Comparison of mean Micro Hardness values between 05 groups using One-way

ANOVA Test

Groups N Mean SD Min Max F P-Value

Group 1 16 50.94 0.31 50.5 51.6

1522.981 <0.001*

Group 2 16 58.83 0.57 57.9 59.8

Group 3 16 61.93 0.46 61.1 63.0

Group 4 16 62.90 0.57 61.7 63.8

Group 5 16 65.65 0.84 64.5 67.3

* - Statistically Significant

Note: Group 1 - EDTA, Group 2 - Chlorine dioxide, Group 3 – QMix

Group 4 - German chamomile, Group 5 – Saline

The table above represents the comparison between the mean microhardness

values according to the Analysis of Variance one-way test. Null hypothesis: There is

no significant difference between the mean microhardness of all 5 groups. Alternate

hypothesis: Since the p value for the ANOVA is less than that of 0.05, it indicates that

Results

42

we should reject the null hypothesis and conclude that there is a significant difference

between the means of all groups.

(Table 2)

Multiple comparison of mean difference in Micro hardness

values between groups using Tukey's HSD Post hoc

Analysis

(I)

Grou

ps

(J)

Grou

ps

Mean

Diff.

(I-J)

95% CI of the

Diff.

P-Value Lower Upper

Group 1 Group 2 -7.89 -8.47 -7.32 <0.001*

Group 3 -10.99 -11.57 -10.42 <0.001*

Group 4 -11.96 -12.54 -11.39 <0.001*

Group 5 -14.72 -15.29 -14.15 <0.001*

Group 2 Group 3 -3.10 -3.67 -2.53 <0.001*

Group 4 -4.07 -4.64 -3.50 <0.001*

Group 5 -6.82 -7.40 -6.25 <0.001*

Group 3 Group 4 -0.97 -1.54 -0.40 <0.001*

Group 5 -3.72 -4.30 -3.15 <0.001*

Group 4 Group 5 -2.75 -3.33 -2.18 <0.001*

* - Statistically Significant

Results

43

The Tukey’s multiple comparisons between the five groups interprets that the

mean microhardness of EDTA differs significantly (p<0.001) from ClO2, QMix, and

Matricaria recutita.

(Graph 1)

All the irrigating solutions decreased the microhardness of root canal dentin

with the exclusion of the control group. The above represented graph shows a

significant difference between the microhardness of all five groups. EDTA exhibited

the highest reduction in root canal dentin microhardness followed by ClO2, QMix,

and German chamomile. The control group with saline showed the least reduction in

the microhardness of root canal dentin.

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

Group 1 Group 2 Group 3 Group 4 Group 5

50.94

58.8361.93 62.90

65.65

Mic

ro H

ard

nes

s V

alu

es

Comparison of mean Micro Hardness

values between 05 groups

Results

44

(Graph 2)

Represented above is a graph showing the average microhardness of dentin

when treated with various irrigants in Vicker’s microhardness numbers on Y-axis.

The tabulated results and the statistical analysis revealed that EDTA showed

the highest degree of reduction in dentin microhardness with a significant difference

of 23.47 % more reduction than Matricaria recutita, which showed the least amount

of dentin microhardness reduction with just a 4.18% difference than the control group

saline. Between ClO2 and QMix, and QMix and Matricaria recutita, there was only

5.26% and 1.51% difference respectively. But EDTA still showed a good 13.41%

higher reduction rate than ClO2.

50.94

58.8361.93 62.90

65.65

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

Group 1 Group 2 Group 3 Group 4 Group 5

Mic

ro H

ard

nes

s V

alu

es

Comparison of mean Micro Hardness

values [with standard errors] between 05

groups

Discussion

45

Long term prognosis of root canal treatment is entirely dependent on the quality

of instrumentation, irrigation, disinfection, and finally the obturation of the root canal

system. A root canal treatment is essentially deemed to be successful, once complete

debridement and disinfection has been achieved. It is a procedure that requires the

removal of irritants from the canal and the periapical tissues carried out in various

ways as the case demands, and includes the instrumentation of the canal, placement

of the medicament and irrigant.30

An irrigant plays a vital role in root canal therapy as they aid not only in

cleaning and shaping, but also in lubricating the canal while using endodontic

instruments. These solutions are required to be used simultaneously with each file

change to avoid unnecessary mishaps like, ledging, canal transportation, improper

cleaning and disinfection till the apex, or instrument separation. The use of chelating

agents as irrigants have helped in opening calcified and narrow canals for better

instrumentation, but at the same time it causes changes in the microstructure of dentin

and the Ca:P ratio which will in turn cause changes in the dentin solubility and

permeability characteristics.

Distilled water was initially used as an irrigant while preparing the canal of the

samples because it has no effect on the microhardness of root canal dentin surface,

thus not considering it as a variable which might affect the results.

This study used single rooted premolar teeth in order to obtain a standardisation

of the teeth and as they were more often extracted for orthodontic purposes, which

increased the chances of its ease of availability for research purposes. Premolars with

Discussion

46

straight canals were important as they are easy to instrument and the procedural errors

like ledging, zipping, apical transportation, and canal blockage can be avoided. To

ensure standardisation, the crown of the selected teeth were removed to maintain a

length of 18mm for each tooth as these served as a reservoir for the solvent to be

present inside the canal for more than the recommended exposure time.

Longitudinal sectioning of the teeth was preferred over cutting it transversally

into discs because as Cruz-Filho et al. in 2013 observed, it provides an accurate

representation of the clinical situations. 37 Ground polishing of the samples eliminated

any surface irregularities and provided a mirror like finish, as the glossy surface

ensures the reflection of light so that the indentation can be clearly visualised when

testing the Vicker’s hardness machine.

A possible limitation of the current study is that the experiments were

performed at room temperature and not body temperature.

An initial rinse with 3% NaOCl can affect the microhardness of root canal

dentin, so it can possible add to the chelating action of the irrigating solutions to be

evaluated; this could be a possible limitation of the present study. De-Deus et al in

2006 limited the contact time of chelator solutions to 5 minutes, stating that this

duration is more realistic in terms of clinical practice. Other researchers like Goldberg

et al in 1982 and Panighi et al in 1992 have suggested extending the application time

to 10 to 15 minutes to obtain optimum results.1

Sodium hypochlorite is known to have an extensive history in medicine and

dentistry and continues to be popular even today. When the Hypochlorous acid

Discussion

47

present in the NaOCl solution comes in contact with the organic tissue it acts as a

solvent and releases chlorine which has antimicrobial action and inhibiting bacterial

enzymes. NaOCl is an efficient organic solvent and has also been known to cause

dentin degeneration because of the dissolution of collagen by the breakdown of bonds

between carbon atoms and disorganisation of the protein primary structure, although

the degeneration of dentin is not as much as EDTA.

EDTA reacts with the calcium ions present in dentin and forms soluble calcium

chelates which decalcifies dentin to a depth of 20-30µm in 5 minutes. When 17%

EDTA is alternated with 5% NaOCl it causes a significant increase in tooth surface

strain which was not seen when used in conjunction with 3% NaOCl. The

consequence of chemical interactions between NaOCl and EDTA results in a loss of

free chlorine, which could explain the inability of their mixture to dissolve soft

tissues. But this was proved otherwise by studies conducted by Grawher et al. in

2003, Irala et al. in 2010, and Saquy et al. in 1994, which reported that the addition of

NaOCl did not alter EDTA’s ability to decalcify human dentin. Nygaard-Ostby in

1957 stated that even though EDTA was forced into the periapical tissues, no

periapical tissue damage could be detected even after 14 months. But on the contrary,

Segura et al. in 1996 showed that the extrusion of even a low concentration of EDTA

solution through the apical constriction can cause irreversible decalcification of

periapical bone and have consequences on the neuro-immunological regulatory

mechanisms.

Chlorhexidine digluconate has been widely used as a disinfectant because of its

excellent antimicrobial activity, but however completely lacks tissue dissolving

Discussion

48

capacity. It has the ability to bind to phosphate present in the structure of

hydroxyapatite which is present in the calcium carbonate complexes in dentin and this

leads to the release of small amounts of calcium from the root canal dentin. CHX also

reacts with EDTA to form a white precipitate, but the clinical significance of this

precipitate is largely unknown. It has been known to cause side effects like contact

dermatitis, desquamative gingivitis, teeth and tongue discolouration.

MTAD was developed as an irrigant by Torabinejad et al. which combined

both chelating and antimicrobial properties. It is a mixture of a detergent, citric acid

which removes the smear layer, making way for the antimicrobial action of

doxycycline in the dentinal tubules. Torabinejad et al. in 2003 stated that a 5 minute

final rinse with MTAD was appropriate after an initial rinse with NaOCl. But another

study by Gopikrishna et al. in 2010 contradicted this by stating that MTAD as a final

rinse reduced the sealing ability of sealers by precipitate formation.

Tetraclean was introduced as a final rinse with a lower concentration of

doxycycline than MTAD. However a study by Giardino et al. in 2007 stated that, only

5.25% NaOCl could result in complete biofilm disgregation, but Tetraclean showed

higher degree of biofilm disgregation than MTAD.38

Herbal products like Triphala, Propolis, Morinda citrifolia juice, green tea etc.

have been later introduced as possible endodontic irrigants with less potential side

effects on the dentin microhardness, even at higher concentrations aimed at removing

smear layer and disinfecting canals.

Discussion

49

Reduction in microhardness could be beneficial under clinical conditions since

it allows rapid preparation and negotiation of tight canals, and the degree of softening

and demineralisation can be an indication that there has been a complete removal of

smear layer which might act as a physical barrier that limits the antibacterial effects

of intracanal medicaments and root canal sealers. Moreover the removal of smear

layer further improves the retention of the root canal filling material. The penetration

of the root canal sealer enables to entomb the residual bacteria that the irrigants failed

to completely remove and also extends the antibacterial effect deep into the dentinal

tubules.37

In the present study, Vicker’s microhardness tester was preferred over Knoop

hardness test due to its suitability and practicality of the test for evaluating surface

changes occurring in the deeper dental hard tissues. This test is also widely accepted

because of its accurate readings and the fact that in this method, just one type of

indentation is used for all types of surface treatments.29

Microhardness testing of materials is usually done by applying small loads and

it is one of the simplest and non-destructive methods of its mechanical

characterisation and is measured as its resistance to the penetration of an indenter that

is harder than the standard to be analysed.32 These tests provide numerical values

which depend on several factors like Young’s modulus of a material, the yield stress

in compression, anisotropy, and thus it cannot be considered a basic property of a

material but rather an indication of the behaviour given the specific conditions of the

penetration test.27

Discussion

50

Based on the results of the present study, all the root canal irrigants reduced the

microhardness of root canal dentin except for the control group, saline. EDTA

showed a microhardness value of 50.94 which was significantly different and 15.48%

higher than ClO2 which showed a hardness value of 58.83. The fact that EDTA acts

efficiently in reduction of dentin microhardness is because of its chelating property

and there are several theories that attempt to explain this chemical reaction.

According to the Crystal Field Theory, the attraction force between the central metal

and the ligands is purely electrostatic. Therefore the attraction force exerted by the

metallic ion is greater than the repulsive force offered by the atoms of the EDTA

molecule. Chelators such as EDTA form a stable complex with the calcium ions in

the dentin and at the same moment the carboxyl ions of the EDTA molecule are

ionized, releasing hydrogen atoms that compete with the calcium ions.37

This result is in accordance with studies conducted by Saleh and Ettman in

1999 which evaluated the effect of EDTA and NaOCl on the microhardness of root

canal dentin and reported that both the solutions decreased the microhardness of root

canal dentin but EDTA irrigation induced more reduction.13 Rama et al., Keyur et al.,

and Anusree et al., in 2014 also stated that EDTA showed the highest amount of

reduction in dentin microhardness as compared to other irrigants used in their

studies.2,39,30 Baldasso et al., in 2017 reported that EDTA showed the highest

reduction in dentin microhardness compared to the other irrigants used in his study.33

Sousa and Silva, Khedmat and Shokouhenijad, and Da Silva et al in 2005 and 2008

respectively, have shown that EDTA facilitates chelation of the inorganic portion of

dentin. Regarding the depths under evaluation, the present study revealed similar

Discussion

51

results about the reduction in microhardness after irrigating with NaOCl followed by

EDTA.1 On the contrary, Kandil et al. in 2014 and Kara et al. in 2015 stated in their

studies that Maleic acid showed higher reduction in dentin microhardness than

EDTA.31,13 They concluded that significant alteration in dentin microhardness after

the irrigation treatment indicates potent direct effects of these chemical solutions on

the components of dentin structure.

The capacity of ClO2 in smear layer removal could be attributed to its low pH

i.e. 4.67 and in the present study, ClO2 also showed high reduction in dentin

microhardness although not more than EDTA. A previous study by Cruz-Filho et al.

in 2011 has suggested that the pH of an irrigant is indirectly proportional to the

demineralisation of root canal dentin. Also ClO2 being a weak acid, it is likely that it

will affect the dentin similar to other organic acids such as Maleic acid or Lactic acid

and it may cause a mineral gradient that could yield itself better to sealer

penetration.22 Another study by Anna et al. in 2013 demonstrated that high purity

ClO2 had the ability to eliminate intracanal biofilm and nearly all the bacteria thus

preventing re-infection in most cases, even more so than NaOCl and CHX when used

as disinfectants.41 Hence, high purity 0.2% chlorine dioxide was used in this study.

QMix being a novel irrigant showed minimum amount of reduction in dentin

microhardness. It is mainly composed of EDTA, CHX, and a surfactant; and the

addition of a surfactant to a chelating agent increases its ability to penetrate the dentin

easily.10 The results of the present study showed that QMix causes a 5% less

reduction in dentin microhardness than ClO2 with a mean microhardness value of

61.93, and a good 17% difference from EDTA. There is only one previous study by

Discussion

52

Aranda-Garcia et al. in 2013 that evaluated the effect of QMix on dentin

microhardness and found that there was no significant difference between QMix and

EDTA. This could be because of the EDTA present in QMix, thus resulting in a

similar reduction in dentin microhardness.31 A study by Tay et al. in 2010 has

reported that QMix completely removed the smear layer and the smear plugs but was

less aggressive than EDTA in that there is less demineralisation of the intact dentin

collagen. The study also showed the long shelf life of QMix, producing a continued

effect on the smear layer as well as its ability to kill resilient strains of Enterococcus

faecalis with induced accelerated aging at 60˚C for 8 weeks, which was the equivalent

of 2 years.42

An herbal product Matricaria recutita or German chamomile has been used

before as an effective natural irrigant which has shown a positive effect on smear

layer removal. In the present study German chamomile showed the least amount of

reduction in dentin microhardness with a good 19% less decrease in microhardness

than EDTA. A study by Hena et al. in 2015 reported that although 2% CHX had a

better effect on smear layer removal and canal disinfection, Matricaria recutita was

better in terms of it being a natural substance with fewer side effects, easy

availability, and a healthier and acceptable host response.15 Its superior medicinal

properties and effective smear layer removal could be attributed to its acidic

components like capric acid, caprylic aid, chlorogenic acid, o-caumaric acid, p-

caumaric acid, and dihydroxybenzoic acid in the extract, which also could be the

reason for the mild reduction in the dentin microhardness.14

Discussion

53

Since there are no studies that showcase the effect of Matricaria recutita on

dentin microhardness, this is the first study to compare and evaluate its difference in

the reduction of dentin microhardness from known irrigants like EDTA, ClO2, and

QMix.

The research focus is increasing more towards natural products for root canal

disinfection owing to their inherent properties like easy availability, cost-

effectiveness, increased shelf life, and low toxicity. Although only few natural

products have shown promising results, more combinations and various

concentrations can improve their efficacy and usage for root canal disinfection in a

bio-friendly way.

Conclusion

54

During cleaning and shaping procedure, the canals should be irrigated using

copious amounts NaOCl. Once the shaping is complete, a rinse with EDTA solution

ensures the removal of smear layer and the decreased application time will minimise

the chelating effect on the root canal dentin. After the smear layer removal procedure,

a final rinse with an antiseptic solution appears beneficial. Since NaOCl has also been

known to reduce the microhardness of root canal dentin, out of the previous irrigating

solutions like CHX, ClO2, MTAD, the herbal product German chamomile appears to

be the most promising agent for use as a final antiseptic irrigant as it produces the

least amount of reduction in the microhardness root canal dentin.

Under the limitations of the present study it was concluded that EDTA showed

the highest degree of reduction in microhardness of root canal dentin surface with an

application time of 5 min followed by ClO2, QMix, and Matricaria recutita

respectively.

Summary

55

Root canal therapy is deemed to be a success when complete debridement

and disinfection of the canal has been established. Although mechanical preparation

of the root canal is a necessity, the process produces a smear layer composed of

organic and inorganic substances. This layer occludes the dentinal orifices and

hinders the penetration of intracanal medicaments and sealers into the dentinal

tubules, thus affecting the fluid tight seal of the root canal system. Chelating agents

were introduced to eliminate microbes, dissolve organic tissue, and remove the smear

layer; but they have been known to cause reduction in microhardness of root canal

dentin.

Novel irrigants like QMix which is a combination of conventional irrigating

solutions like EDTA and CHX have been introduced, that produce the same effect as

conventional irrigants but with a lesser degree of decalcifying detrimental effects

affecting the root canal dentin. Irrigating solutions play a key role in the cleaning and

shaping of the root canal system by producing a chelating effect on the root canal

dentin as well as deliver antimicrobial properties into the root canal.

This study aimed to evaluate and compare the effect of novel root canal

irrigants like QMix and Matricaria recutita with conventional irrigants like EDTA and

ClO2, on the microhardness of root canal dentin. The null hypothesis is that there is

no difference between EDTA, QMix, ClO2, and Matricaria recutita on the

microhardness of root canal dentin.

Forty single rooted premolar teeth were selected in this study and stored in

hydrogen peroxide for a week and then normal saline until it was used. The root

Summary

55

canals were prepared using Protaper Universal files and distilled water for irrigation

to avoid unnecessary changes to the dentinal structure. Initial rinsing was done for 5

minutes using NaOCl and then the samples were treated with EDTA, ClO2, QMix,

and Matricaria recutita for another 5 minutes. The teeth were decoronated to ensure

standardization of 18mm and split longitudinally before embedding in acrylic resin.

The teeth were then randomly divided into 5 groups with 16 samples each. Group I-

EDTA, Group II- ClO2, Group III- QMix, Group IV- Matricaria recutita, Group V-

Saline.

Each sample was then subjected to a load of 100g for 15 seconds on the

Vicker’s hardness tester to obtain 3 readings for each sample in each group. The

results were tabulated and statistically analysed to obtain a mean result which

determined which of the irrigating solutions caused the most reduction in root canal

dentin microhardness. And it was concluded that EDTA caused the most reduction in

dentin microhardness followed by ClO2, QMix, and the herbal product Matricaria

recutita.

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Annexure

62

Annexure

63

Annexure

62

Annexure

63

Annexure

64

Annexure

65

Microhardness values for samples with EDTA

Samples Reading 1 Reading 2 Reading 3

1 51.26 50.09 50.93

2 52.03 51.87 50.97

3 51.01 51.86 50.93

4 51.11 51.00 50.34

5 50.67 52.90 50.89

6 51.23 50.68 50.67

7 50.14 50.77 51.19

8 50.32 51.05 51.07

9 51.24 51.78 50.40

10 50.87 50.43 50.22

11 51.97 50.67 50.37

12 51.03 50.22 51.08

13 50.97 50.39 50.27

14 52.06 50.76 50.33

15 50.95 51.02 50.16

16 51.30 50.98 50.45

Microhardness values of samples with Chlorine dioxide


1 59.26 60.09 59.93

2 58.23 59.07 58.17

3 57.71 58.36 59.04

4 56.91 58.20 59.14

5 59.67 58.93 57.99

6 57.97 56.08 59.67

7 59.14 58.77 59.29

8 58.39 61.05 58.27

9 60.24 59.00 59.47

10 58.87 58.13 60.12

11 59.07 57.00 58.67

12 61.03 58.92 55.08

13 58.27 59.34 59.37

14 59.76 57.92 58.13

15 58.95 58.32 59.70

16 58.73 60.28 60.15

Annexure

66

Microhardness values for samples with QMix


1 61.29 60.99 60.93

2 62.05 61.87 61.97

3 61.29 62.86 62.03

4 61.91 61.90 61.94

5 60.90 62.70 62.19

6 62.23 61.88 61.97

7 60.94 60.77 61.99

8 61.92 61.55 62.07

9 61.97 62.78 61.49

10 62.87 63.30 62.92

11 61.97 61.97 61.95

12 62.73 61.92 61.98

13 62.55 62.32 61.77

14 62.05 62.76 62.06

15 60.95 61.92 60.96

16 62.39 60.98 61.95

Microhardness values for German chamomile


1 63.69 63.88 63.94

2 62.97 61.99 60.91

3 63.39 62.39 64.00

4 64.01 63.46 62.01

5 63.92 63.00 62.57

6 63.99 63.04 62.89

7 61.97 62.94 63.90

8 62.99 61.99 60.05

9 62.69 62.79 65.33

10 63.00 63.08 62.01

11 62.79 62.78 62.45

12 61.09 61.90 63.77

13 61.99 62.97 63.86

14 62.87 64.09 62.40

15 64.01 63.42 62.04

16 63.99 62.98 60.96

Annexure

67

Control group - Saline


1 65.98 66.30 67.01

2 66.01 65.56 64.08

3 66.35 67.00 66.99

4 64.86 65.02 63.98

5 65.55 66.16 64.98

6 66.78 65.03 65.00

7 68.80 67.05 66.08

8 66.66 65.06 65.00

9 65.01 67.00 66.66

10 61.98 65.55 65.99

11 64.35 65.08 64.07

12 66.80 67.08 65.55

13 67.00 65.55 66.00

14 65.05 64.55 65.55

15 66.11 63.99 64.98

16 65.88 64.68 65.60

Annexure

68

Annexure

62

Microhardness values for samples with EDTA


Microhardness values of samples with Chlorine dioxide


Annexure

63

Microhardness values for samples with QMix


Microhardness values for German chamomile


Annexure

64

Control group - Saline


Scanned by CamScanner

Photographs

39

Teeth samples Pro Taper Universal Files

Endomotor Tooth embedded in resin

Saline

Sodium Hypochlorite

Photographs

40

EDTA

Matricaria recutita seeds

Chlorine dioxide

Application of irrigants

Vicker’s microhardness test

QMix 2in1 solution

Photographs

41

“an evaluation of the effect of different irrigating

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