Calmix final print web

A NEW IMPROVED CALCIUM HYDROXIDE MEDICAMENT PASTE FOR ENDODONTICS

Better dissolution of calcium hydroxide

Higher ionization & pH achieved (CALMIX recorded > 14)

High pH

High antimicrobial potential: Effective against E.faecalis & C.albicans

Greater diffusion of hydroxyl ions

Stimulates hard tissue formation

Contains Ziconium Dioxide

CALMIX is more radiopaque

Water free & no methyl cellulose

Will not dry out

Ergonomic syringes, capillary tips & microtips

Easy to use, no mixing

2

In any water-based media (liquid or paste), the common ion effect from OH ions already present in water limits dissolution of calcium hydroxide powder1. changed the vehicle to something other than water.

This gives greater solubility, avoids the common ion effect, gives greater release, and allows higher pH values to be achieved2. uses a special co-polymer of PEG400 and PEG4000 designed to give optimal calcium hydroxide dissolution and release.

A titration study using hydrochloric acid challenge revealed that OH ion release from Ca(OH)2 in PEG400 was significantly higher than for Ca(OH)2 in a saturated solution in water (P<0.01)3.

Athanassiadis B, Abbott PV, George N, Walsh LJ. In vitro study of the inactivation by dentine antimicrobial endodontic medicaments and their bases. Australian Dental Journal 2010; 55 (???)

1

Walsh LJ, Athanassiadis B. Non-aqueous solvents influence pH of calcium hydroxide products.

IADR ANZ 2014.

2

Walsh LJ, Athanassiadis B. Influence of PEG400 vehicle on calcium hydroxide medicaments. IADR 2016.

3

3

High pH is achieved by medicaments.

when compared with other

The pH of commercial endodontic pastes measured using two different electrodes Pulpdent

Calasept Plus

Odontocide

Calmix

Ionode electrode

12.706 c (0.006)

12.662 c (0.017)

13.170 b (0.025)

14.996 a (0.010)

Solvotrode electrode

12.862 b (0.008)

12.710 b (0.012)

13.062 b (0.450)

15.162 a (0.108)

pH value stated by manufacturer

> 12.0

12.4

12.5

> 13.5

Data show the mean and SD from five independent experiments.

Letters indicate statistical groupings across each row ranked from highest pH (a) to lowest pH (c). Information on product pH stated by the manufacturer was obtained from material safety data sheets and manufacturer websites.

maintains high pH over longer periods of time.

Maximum pH values obtained and Time to maximum pH in different levels of dentine Max pH - inner dentine

Max pH - outer dentine

Time to max pH - inner dentine

Time to max pH - outer dentine

Control (no medicaments)

7.45

CALMIX (no smear layer)

11.27

7.46

2 days

10 weeks

8.92

1 week

CALMIX & Ledermix 50/50 (no smear layer)

4 weeks

9.51

8.98

4 weeks

10 weeks

Calasept & Ledermix 50/50 (no smear layer)

9.39

8.58

4 weeks

10 weeks

Pulpdent

9.96

8.62

2 weeks

12 weeks

Material

Calcium hydroxide diffusion through radicular dentine

pH and total hydroxyl ion diffusion from the 50:50 mixtures was lower and slower than when Ca(OH)2 was used alone

4

is more effective against fungi than all other calcium hydroxide pastes.

Antimicrobial testing - Lower MBCs means greater effectiveness MBC (mg/ml) Medicaments

E.faecalis

Candida albicans

PEG 400/3350 base

No effect

No effect

7.80

7.80

Calasept

7.80

125.00

Pulpdent

7.80

62.5

Odontocide

31.25

31.25

Results are the mean of three replicates. Assays done by a commercial microbiology lab using blinded samples.

Least effective on EF

CALMIX is better against fungi than all other calcium hydroxide pastes

Greater effectiveness against candida species Time kill study - Log reduction following 1 hour contact time with medicament Medicaments

E.faecalis

Candida albicans

PEG paste only

0.1, 0.0

0.5, 0.3

Ledermix

No effect

0.1, 0

Pulpdent

> 5.6, > 5.6

2.9, 2.7

> 5.6, > 5.6

> 4.1, > 4.2

Reduction factor is approx.

400,000 fold from 1 hr contact time

Greater effectiveness against Candida species compared to Pulpdent and Ledermix

5

Despite a higher pH, Cytotoxicity Scale

is less cytotoxic (tissue injury)

Interpretation

Condition of cell cultures

0

Non cytotoxic

≤ 30% cells are round, loosely attached, only slight growth inhibition observable

1

Slightly cytotoxic

≥ 30% cells are round, loosely attached, only slight growth inhibition observable

2

Mildly cytotoxic

≥ 50% cells are round, not more than 50% growth inhibition observable

3

Moderately cytotoxic

≥ 70% cells are lysed but more than 50% growth inhibition observable

4

Severely cytotoxic

Complete destruction of the cell monolayers

The cell line used to perform this investigation was Vero cells, obtained from the ATCC. This is a standard cell line for use in cytotoxicity testing

Treatment Group

Cytotoxicity Scale (Individual Results) Pulpdent

Ultracal

Neat (100%) Extract

4, 4, 4

4, 4, 4

4, 4, 4

50% Extract

4, 4, 4

4, 4, 4

4, 4, 4

40% Extract

4, 4, 4

4, 4, 4

4, 4, 4

30% Extract

4, 4, 4

4, 4, 4

3, 3, 3

20% Extract

2, 2, 2

2, 2, 2

0, 0, 0

10% Extract

0, 0, 0

0, 0, 0

0, 0, 0

5% Extract

0, 0, 0

0, 0, 0

0, 0, 0

Negative Control

0, 0, 0

Medium Control

0, 0, 0

Blank Control

0, 0, 0

Positive Control

4, 4, 4

The level of cytotoxicity for the samples Pulpdent and Ultracal also decreased gradually with mild cytotoxicity at 20% dilution and no cytotoxicity detected at 10% and lower dilutions. Thus, the test sample “CALMIX” has proved to be less cytotoxic than the reference samples “Pulpdent” or “Utracal” when tested as described above as per ISO 10993-5 (2009).

6

Grey scale value (higher = more radiopaque)

Comparison

Mean Difference

q

P Value

vs Calasept

5.020

24.847

P < 0.001

vs Pulpdent

8.590

40.834

P < 0.001

vs Ultracal

37.170

177.09

P < 0.001

vs Odontocide

70.310

327.96

P < 0.001

7

KEY POINTS Higher pH than all existing Ca(OH)2 medicaments Greater antimicrobial activity within the root canal Less inflamation to outer tissues than Pulpdent & Ultracal Wonâ&#x20AC;&#x2122;t dry out on pad or in syringe Easily picked up with spiral or placed in canal with fine tips

8

ydroxide chieved ods and e glycol hydroxyl Results: es made saline). icament ions will

butes of dotoxins, oxide in g/100 OC with sodium ssues for

dodontic have a tertown, a wateres, since dodontic calcium xide into can be act as a ounding

C LCI N LIN C IACLA L

The Theinfluence influenceofofaqueous aqueousand andPEG PEG400 400 solvent solventvehicles vehiclesononhydroxyl hydroxylion ionrelease release from fromcalcium calciumhydroxide hydroxidemedicaments medicaments 1 1 1,2 1,2 1 1 Yu-Yao Yu-Yao Teoh, Teoh, Basil Basil Athanassiadis Athanassiadis andand Laurence Laurence J. Walsh J. Walsh

Abstract Abstract Objectives: Objectives: Calcium Calcium hydroxide hydroxide pastes pastes havehave beenbeen usedused in endodontics in endodontics sincesince 1947.

1947.

MostMost current current calcium calcium hydroxide hydroxide endodontic endodontic pastes pastes use use water water as the as vehicle, the vehicle, which which limitslimits the dissolution the dissolution of calcium of calcium hydroxide hydroxide which which can can be achieved be achieved andand thereby thereby the maximal the maximal hydroxyl hydroxyl ion ion release release which which can can be achieved within the root canal system.

Methods andand be achieved within the root canal system.

Methods Materials: Materials: compared the ionic properties of calcium hydroxide solutions in water or polyethylene glycol This This studystudy compared the ionic properties of calcium hydroxide solutions in water or polyethylene glycol (PEG), the ionic properties of commercial endodontic medicaments made water or PEG, finally hydroxyl (PEG), thenthen the ionic properties of commercial endodontic medicaments made usingusing water or PEG, and and finally hydroxyl ion release radicular dentine in vitro using a new model colour change in anthocyanin stained roots.

Results: ion release into into radicular dentine in vitro using a new model withwith colour change in anthocyanin stained roots.

Results: PEGPEG 400400 as aas solvent gavegave a higher pH and moremore bio-available a solvent a higher pH and bio-available hydroxyl water.

Commercial pastes made hydroxyl ionsions thanthan water.

Commercial pastes made using as solvent the solvent showed a greater pH than those formulated aqueous vehicles (water or saline). using PEGPEG 400400 as the showed a greater pH than those formulated withwith aqueous vehicles (water or saline).

Hydroxyl ion penetration under physiological conditions greater when as medicament the medicament Hydroxyl ion penetration into into rootsroots under physiological conditions waswas greater when PEGPEG waswas usedused as the mixtures of PEG water.

Clinical significance: Enhanced basebase thanthan mixtures of PEG andand water.

Clinical significance: Enhanced availability andand release of hydroxyl ionsions will will availability release of hydroxyl enhance antimicrobial enhance antimicrobial actions of calcium hydroxide when the active agent in endodontic medicaments. actions of calcium hydroxide when it is ittheis active agent in endodontic medicaments.

Keywords: Calcium hydroxide, 400, non-aqueous solvents, Keywords: Calcium hydroxide, PEGPEG 400, non-aqueous solvents, pH pH Short pHcalcium of calcium hydroxide medicaments Short Title:Title: pH of hydroxide medicaments

Introduction Introduction Hydroxyl Hydroxyl ion ion release release fromfrom calcium calcium hydroxide hydroxide is responsible is responsible for the for key the key attributes attributes of of broad broad spectrum spectrum antimicrobial antimicrobial activity, activity, penetration penetration into into biofilms, biofilms, inhibition inhibition of endotoxins, of endotoxins, 1,2 1,2 and and dissolution of organic tissues. dissolution of organic tissues.

The The maximum maximum solubility solubility of calcium of calcium hydroxide hydroxide in in water water at 25 at OC 25 OC is only is only 0.159 0.159 g/100mL g/100mL (0.16%), (0.16%), andand this reduces this reduces to 0.140 to 0.140 g/100 g/100 mL (0.14%) mL (0.14%) at 40 at OC, 40 OC, withwith an accompanying an accompanying decrease decrease of 0.033 of 0.033 pH unit/OC pH unit/OC withwith 3 3 increasing increasing temperature. temperature.

Calcium Calcium hydroxide hydroxide is much is much less less soluble soluble in water in water thanthan sodium sodium 1 The1 The University University of Queensland, of Queensland, hydroxide hydroxide or potassium or potassium hydroxide, hydroxide, however however bothboth the latter the latter too caustic too caustic to soft tissues for for to soft tissues Brisbane, Brisbane, Australia Australia 4 4 clinical use.use. clinical 2 2 To gain To gain the greatest the greatest antimicrobial antimicrobial actions, actions, release release of hydroxyl of hydroxyl ionsions fromfrom endodontic endodontic Private Private Dental Dental Practice, Practice, Brisbane, Brisbane, Australia Australia pastes pastes mustmust be optimized. be optimized.

Traditional Traditional water-based water-based calcium calcium hydroxide hydroxide pastes pastes havehave a a TM TM longlong history history of clinical of clinical use,use, withwith Pulpdent Pulpdent paste paste (Pulpdent (Pulpdent Corporation, Corporation, Watertown, Watertown, Corresponding Corresponding author: author: Massachusetts, Massachusetts, USA)USA) having having beenbeen on the on market the market sincesince 1947.

1947.

In products In products withwith a watera waterProfessor Professor Laurence Laurence J. Walsh J. Walsh based based vehicle vehicle (e.g.(e.g. distilled distilled water or saline), the common ion ion effect operates, sincesince water or saline), the common effect operates, The The University University of Queensland of Queensland water water already contains some hydroxyl calcium hydroxide endodontic already contains some free free hydroxyl ions.ions.

MostMost calcium hydroxide endodontic School School of Dentistry of Dentistry pastes which water-based calcium hydroxide contain a large excess of calcium pastes which are are water-based calcium hydroxide contain a large excess of calcium Health Centre, UQ UQ OralOral Health Centre, 2 2 hydroxide, beyond which be dissolved. hydroxide, wellwell beyond that that which can can be dissolved.

Placing Placing calcium calcium hydroxide hydroxide into into Herston Road, Herston 288288 Herston Road, Herston water water in amounts in amounts above above the the solubility solubility limitlimit will will not not elevate elevate the the pH pH which which can can be be 4006 Australia QLDQLD 4006 Australia achieved, achieved, sincesince no further no further material can can dissolve.

The The undissolved material will will act as material dissolve. undissolved material actaas a E: l.walsh@uq.edu.au E: l.walsh@uq.edu.au fillerfiller or thickener or thickener in the in the paste, paste, andand as hydroxyl as hydroxyl ionsions diffuse diffuse into into the the surrounding surrounding T: + T:61-7-33658160 + 61-7-33658160 environment, environment, it can it can alsoalso act as actaasreservoir. a reservoir.

F: +F:61-733658199 + 61-733658199 42 42 INTERNATIONAL INTERNATIONAL DENTISTRY DENTISTRY â&#x20AC;&#x201C; AUSTRALASIAN â&#x20AC;&#x201C; AUSTRALASIAN EDITION EDITION VOL.VOL. 11, NO. 11, NO. 4 4

C L ICNLI ICNAI C L AL

TableTable 1. Relationships 1. Relationships between between ion concentrations, ion concentrations, pH, pOH pH, pOH and millivoltage and millivoltage pH pH

H+ H+ concentration concentration (mol/l) (mol/l)

pOH pOH

Table 1

OH- OHconcentration concentration (mol/l) (mol/l)

mV mV

14

14

1x10-14 1x10-14

+414.12 +414.12

0.1 0.1

13

13

-13

1x10 1x10

+354.96 +354.96

1x10-41x10-4

10

10

1x10-10 1x10-10

+177.48 +177.48

-7 1x 101x 10-7

7

7

-7 1x 101x 10-7

9

-9 1x 101x 10-9

5

5

-5 1x 101x 10-5

-118.32 -118.32

12

12

-12 1x 101x 10-12

2

2

0.01 0.01

-295.80 -295.80

14

14

-14 1x 101x 10-14

0

0

0

0

1

1

4

4

7

7

9

1

1

-13

1

1

0

0

-414.12 -414.12

Mathematical Mathematical relationships relationships seen in seen pure in water pure water at 25oC at 25oC between between pH, pOH, pH, pOH, hydroxyl hydroxyl ion concentrations ion concentrations and millivoltage and millivoltage recordings recordings for conventional for conventional pH electrodes pH electrodes with KCl withelectrolyte.

KCl electrolyte.

Mathem recordin

There There is a long of theof use non-aqueous liquidsliquids is a history long history theofuse of non-aqueous in in measurement measurement of nonof - non aqueous - aqueous solutions, solutions, using using reference reference endodontic pastes, the mid-1970’s endodontic pastes, goinggoing back back to thetomid-1970’s when when electrolytes electrolytes such as such lithium as lithium chloride chloride 2 mol/l 2 mol/l in ethanol in ethanol or or components components such as polyethylene glycolglycol (PEG)(PEG) 400 were tetraethylammonium tetraethylammonium bromide bromide (TEABr) (TEABr) 0.4 mol/l 0.4 mol/l in ethylene in ethylene such as polyethylene 400 were addedadded to alter to improve the clinical handling glycol), for acidic for acidic and alkaline and alkaline materials, materials, respectively. respectively.

UsingUsing to the alterviscosity the viscosity to improve the clinical handlingglycol), of theofpaste.

As anAsexample, the 1976 formulation of a of asuch such metersmeters and electrodes, and electrodes, assessments assessments of hydroxyl of hydroxyl ion ion the paste. an example, the 1976 formulation calcium hydroxide paste paste (Calen) included both PEG concentration (pOH)(pOH) and pH andcan pHbe can made be made for aqueous calcium hydroxide (Calen) included both 400 PEG 400concentration for aqueous and and and colophony resin, resin, with the beingbeing a thickening non-aqueous materials. are mathematical relationships and colophony withlatter the latter a thickeningnon-aqueous materials.

There There are mathematical relationships agent.agent.

OtherOther viscosity modifiers used in calcium hydroxide viscosity modifiers used in calcium hydroxide between pOH,pOH, pH and (Table(Table 1). When between pH millivoltage and millivoltage 1). When 1 pastespastes have have included glycerol and propylene considering the ionic of non-aqueous solvents, one one considering the properties ionic properties of non-aqueous solvents, included glycerol and propylene glycol.glycol.

It is 1 It is has tohas consider dissociation of theofsolvent and the to consider dissociation the solvent andslower the slower now recognised now recognised that such thatnon-aqueous liquidsliquids are not merely such non-aqueous are not merely response time of non-aqueous electrodes, whichwhich require response time of non-aqueous electrodes, require passive agents, but may contribute to thetochemical passive agents, but actively may actively contribute the chemical extended measurement times.times.

Thus, Thus, the first theof study extended measurement thepart firstofpart the study and biological properties of theof paste, by enhancing and biological properties the paste, by enhancing assessed ionic ionic properties of calcium hydroxide assessed properties of calcium hydroxide in different dissolution of calcium hydroxide and release of hydroxyl dissolution of calcium hydroxide and release of hydroxyl in different solvents, the second the properties ionic properties of calcium ions. PEG used contemporary endodontic pastespastes ions. 400 PEG is 400 is in used in contemporary endodontic solvents, the second the ionic of calcium hydroxide hydroxide whichwhich contain calcium hydroxide, calcium hydroxide plus plusmedicaments contain calcium hydroxide, calcium hydroxide with different compositions, and the medicaments with different compositions, andthird the the third the ibuprofen, or antibiotics ibuprofen, or antibiotics and corticosteroids. diffusion of hydroxyl ions into extracted teeth. teeth. diffusion of hydroxyl ionsroots into of roots of extracted and corticosteroids.

Theofaim the present was therefore The aim theofpresent study study was therefore to assess the the to assess chemical properties of PEG 400 400 when when used used as a as nonchemical properties of PEG a nonMaterials Materials and Methods and Methods aqueous solvent for calcium hydroxide, with awith particular aqueous solvent for calcium hydroxide, a particular Part 1.

Part Ionic 1. Ionic properties properties of solutions of solutions emphasis on theonrelease of hydroxyl ions. Such can can emphasis the release of hydroxyl ions. release Such release A research A research gradegrade pH meter pH meter (model (model HI 4222, HI 4222, Hanna Hanna be considered in terms of theofpOH and pH WhileWhile be considered in terms the pOH andscales. pH scales.

Instruments, Instruments, Woodsocket, Woodsocket, RhodeRhode Island,Island, USA) USA) was used was for used for originally theorized

by Sorensen for water-based solutions, originally theorized

by Sorensen for water-based solutions, all measurements. all measurements.

The meter The meter was calibrated was calibrated using using four four it is now that the it is well nowknown well known thatpHthescale pH scale canbe also be as used as standard aqueous pH values of 4, 10,13 and 13 standard aqueous buffersbuffers at pHatvalues of 4, 7, 10,7,and can also used a measure the acid-base properties of non-aqueous (ACR (ACR Chemical Reagents, Moorooka, Queensland, a measure of theof acid-base properties of non-aqueous Chemical Reagents, Moorooka, Queensland, 5,6 5,6 Two different electrodes were were used to measure the the solutions. solutions.

Australia).

Two different electrodes used to measure This isThis done is done typically typically using using research-grade research-grade pH pHAustralia). medicaments and different calcium hydroxide/PEG in medicaments and different calcium hydroxide/PEG metersmeters that display that display both pH both(from pH (from -2.00-2.00 to +20.00) to +20.00) and andpH inpH the firstthebeing a conventional electrode (model(model 400 mixtures, first being a conventional electrode millivolts, millivolts, and which and which are fitted are fitted with either with either conventional conventional400 mixtures, Ionode, Tennyson, Queensland, Australia), while while the the IJ-44C, Ionode, Tennyson, Queensland, Australia), electrodes electrodes or electrodes or electrodes specifically specifically designed designed for the for theIJ-44C,

There endodo compon added t of the p calcium and co agent. O pastes h now rec passive and bio dissoluti ions. PE which c ibuprofe The a chemica aqueou emphas be cons originall it is now a meas solutions meters t millivolts electrod

VOL. 11, VOL. NO. 11,4 NO. INTERNATIONAL 4 INTERNATIONAL DENTISTRY DENTISTRY – AUSTRALASIAN – AUSTRALASIAN EDITION EDITION 43 43

T ETOEHO H / A/ TAHTAHNAANSASSI A S IDAI D S I /S W / A WLAS LHS H

Table Table 2. Components 2. Components of commercial of commercial calcium calcium hydroxide hydroxide pastes pastes

de

Pulpdent Pulpdent

Calasept Calasept PlusPlus

Active Active ingredients ingredients

39-42% 39-42% Ca(OH) Ca(OH) 2 2

41.07 41.07 % Ca(OH) % Ca(OH) 2 2

37.5% 37.5% Ca(OH) Ca(OH) 2 2

20%20% Ca(OH) Ca(OH) 2 2 7% Ibuprofen 7% Ibuprofen

Vehicle Vehicle

Water Water

Isotonic Isotonic saline saline

45.5% 45.5% PEGPEG 400400

61%61% PEGPEG 400400 5% Water 5% Water

00

Thickener Thickener

Methyl Methyl cellulose cellulose

Nil Nil

PEGPEG 4000 4000

3000 2.3%2.3% PEGPEG 3000

phate

Radiopaque Radiopaque agent agent

Barium Barium sulphate sulphate

8.33% 8.33% Barium Barium sulphate sulphate

14%14% Zirconium Zirconium dioxide dioxide

2% Barium sulphate 2% Barium sulphate

0

CalMix CalMix

Odontocide Odontocide

al safety

Components Components are are shown shown in percentages in percentages by weight. by weight.

Information Information on product on product composition composition waswas obtained obtained fromfrom material material safety safety datadata sheets sheets and and manufacturer manufacturer websites. websites.

d then alysis of ets met

second waswas second designed specifically for nonaqueous solvents experiments designed specifically for nonaqueous solvents experiments werewere assessed assessed for for normality, normality, andand thenthen (Solvotrode, Metrohm Instruments, Gladesville, NSW, differences (Solvotrode, Metrohm Instruments, Gladesville, NSW, differences analyzed analyzed using using Student’s Student’s t test, t test, or analysis or analysis of of Australia), contained lithium chloride in ethanol Australia), andand contained 2% 2% lithium chloride in ethanol as as variance variance withwith Bonferroni Bonferroni post-tests. post-tests.

DataData sets sets met met the reference electrolyte.

As instructed by latter the latter electrode requirements the reference electrolyte.

As instructed by the electrode requirements for parametric for parametric statistical statistical comparisons. comparisons. manufacturer, for testing alkaline materials the lithium chloride manufacturer, for testing alkaline materials the lithium chloride in ethanol electrolyte in ethanol electrolyte waswas removed andand replaced withwith a a PartPart removed replaced 2. Ionic 2. Ionic properties properties of medicaments of medicaments solution of TEABr in ethylene glycol priorprior to use.

When the the The The solution of TEABr in ethylene glycol to use.

When pH of pHfour of four commercial commercial calcium calcium hydroxide hydroxide medicaments medicaments electrodes in use stored in their waswas electrodes werewere not not in use theythey werewere stored in their measured.

TwoTwo of the medicaments werewere measured. of commercial the commercial medicaments TM TM recommended storage solutions in water for the water recommended storage solutions (2% (2% KCl KCl in water for the based (Pulpdent water based (Pulpdent paste, paste, Pulpdent Pulpdent Corporation, Corporation, Ionode electrode, TEABr in ethylene glycol for the Watertown, Ionode electrode, andand TEABr in ethylene glycol for the Watertown, Massachusetts, Massachusetts, USA, USA, andand Calasept Calasept PlusTM PlusTM , , Solvotrode electrode).

Solvotrode electrode).

Nordiska Nordiska Dental, Dental, Angelholm, Angelholm, Sweden) Sweden) andand two two usedused PEGPEG of various concentrations of calcium hydroxide 400400 The The pH pH of various concentrations of calcium hydroxide as their as their basebase (OdontocideTM, (OdontocideTM, Australian Australian Dental Dental (analytical grade, Chem-Supply, Gillman, South Australia, Manufacturing, (analytical grade, Chem-Supply, Gillman, South Australia, Manufacturing, Kenmore Kenmore Hills,Hills, Queensland, Queensland, Australia, Australia, andand Australia) placed Australia) placed in ultrapure water or polyethylene glycol CalmixTM, Ozdent, Ozdent, Castle Castle Hill,Hill, NSW, NSW, Australia).

Australia).

The The in ultrapure water or polyethylene glycol CalmixTM, 400400 (Ace(Ace Chemical Company, Camden Park,Park, South composition of these of these medicaments medicaments is given is given in Table 2. 2.

Chemical Company, Camden South composition in Table Australia, the the two two PEGPEG 400-based commercial medicaments Australia, Australia) measured, at final concentrations 400-based commercial medicaments Australia) waswas measured, at final concentrations of of For For Odontocide) between 10%, 40%.

Calcium hydroxide powder (Calmix, Odontocide) between 60 and minutes 5%,5%, 10%, 20%20% andand 40%.

Calcium hydroxide powder waswas (Calmix, 60 and 140140 minutes waswas added to PEG in disposable plastic tubes 15 mm required required before a stable value reached, whilst the water added to PEG 400400 in disposable plastic clearclear tubes 15 mm before a stable value waswas reached, whilst the water diameter mixed vigorously using based medicaments (Pulpdent, Calasept diameter andand 95 95 mm mm high,high, andand mixed vigorously using a a based medicaments (Pulpdent, Calasept plus)plus) tooktook onlyonly 10 10 metal spatula for seconds 20 seconds to achieve a uniform minutes to reach a stable value.

Pulpdent metal spatula for 20 to achieve a uniform mix.mix.

The The minutes to reach a stable pH pH value.

For For Pulpdent andand Solvotrode electrode inserted Calasept (aqueous solvent), between measurements Solvotrode electrode waswas inserted into into this this mixture and and the the Calasept Plus Plus (aqueous solvent), between measurements the the mixture electrode washed by vigorous stirring in three separate pH recorded at room temperature (23OC) every 5 minutes waswas washed by vigorous stirring in three separate pH recorded at room temperature (23OC) every 5 minutes electrode lotsdistilled until until a stable lots of water for 2forminutes eacheach to remove any any paste a stable measurement reached. measurement waswas reached. of distilled water 2 minutes to remove paste andand to reduce values of five separate samples for each of the on the The The pH pH values of five separate samples for each of the on electrode, the electrode, to reduce the final pH registered on the the final pH registered on the different concentration of calcium hydroxide/PEG different concentration of calcium hydroxide/PEG 400400 electrode electrode to approximately pHto7,ensure to ensure a neutral reading to approximately pH 7, a neutral reading mixtures recorded. all pH measurements, values at the mixtures werewere recorded.

For For all pH measurements, values at electrode the electrode tip prior to further readings.

For Calmix tip prior to further readings.

For Calmix and and tracked became consistent.

For the Odontocide werewere tracked overover timetime until until theythey became consistent.

For the Odontocide solvent), the same regimen followed, (PEG(PEG solvent), the same regimen waswas followed, different mixtures of calcium hydroxide in PEG 400, between but the different mixtures of calcium hydroxide in PEG 400, between but electrodes the electrodes placed in their relevant storage werewere thenthen placed in their relevant storage 60 and 140140 minutes waswas required before a stable value waswas solutions solutions a minimum period of five for afor minimum period of five minutes to replenish the the 60 and minutes required before a stable value minutes to replenish reached.

Recorded values for pH tip after which it was washed in distilled water. reached.

Recorded values for pH independent electrode electrode tip after which it was washed in distilled water. fromfrom five five independent

caments nts were oration, lusTM , ed PEG Dental lia, and a). The 2. caments tes was he water only 10 ent and ments the eparate ny paste d on the reading mix and ollowed, storage enish the water.

44 44 INTERNATIONAL INTERNATIONAL DENTISTRY DENTISTRY – AUSTRALASIAN – AUSTRALASIAN EDITION EDITION VOL.VOL. 11, NO. 11, NO. 4 4

%

9)0.089)

18) a (0.118)

row, each row,

alkaline e pH pH entwo the two ode waysalways g ranking of of was lmix, Calmix, ,both with both erials n materials node the Ionode erials n materials pdent or Pulpdent rode Solvotrode other and other ), there but there hree astes.pastes. were rodes were igher eing higher ween gs between ant r allfor all ewhere the the

ntains ch contains ofen, ibuprofen, 5% cium calcium H esvalues for for ide PEGin PEG ed 40% for 40%

neeks the in the changes es in in eated with with

TTEEOOHH // AATTHHAANNAASSSSI IAADDI ISS // W WAALLSSHH

Part 3. 3.

Hydroxyl release hydroxide into radicular dentine Table The pH ion of calcium in PEG 400 The experimental model involved tracking changes in the Ca(OH) 5% 20 % 40% 2 colour of anthocyanin dye in stained roots kept at 10% concentration physiological conditions. Anthocyanin is the pH indicator found in red cabbage, and it undergoes multiple colour Ionode d (0.115)range,13.344 13.938 b (0.066) 14.75 a (0.089) changes with pH over 12.918 the alkaline with c (0.117) electrode corresponding increases in both the red and green channels as the colour changes. A series of buffers from pH 8.0 to Solvotrode d (0.083) 13.598 14.298 b (0.078) 14.940 a (0.118) 13.0electrode were prepared to serve13.260 as a reference for changes in c (0.063) pixel data (Fig. 1). Extracted human permanent teeth were collected from an oral surgery clinic with the approval of the Data show the mean and SD from five independent experiments. Letters indicate statistical groupings across each row, institutional ethics committee. A total of 15 single-canal teeth ranked from highest pH (a) to lowest pH (d). free from intrinsic discolouration or translucency were selected, then the crowns removed at the cemento-enamel junction and the root face smoothened to form a flat coronal collected usingabrasive the histogram gaveofred andcanals green surface using discs.tool, Afterwhich patency root Ionic properties of medicaments valuesconfirmed for the selected on aK-file scale (Dentsply with 256 points.

The was using area a #8 Maillefer, Medicaments based on PEG400 had a more alkaline pH extent of change baseline was calculated for therotary three Switzerland), theyfrom were then prepared with NiTi (Table 4). Differences in the readings between the two replicates for each group, and analysed using GraphPad instruments (ProTaper Next, Dentsply Maillefer, Switzerland) electrodes were consistent, with the TEABr electrode always applying non-parametric analyses of for toPrism™ size X3software with variable taper and an apical preparation being higher by 0.05-0.16 pH units. The ranking of repeated well aswere intra-group comparisons.

ISO #30.samples The rootascanals irrigated alternately with medicaments from most alkaline to least alkaline was Calmix, sodium hypochlorite and ethylenediaminetetraacetic acid Odontocide, Pulpdent and then Calasept Plus, with both Resultsusing syringes with side-vented needles. After a final electrodes. Measured pH differences between materials (EDTA) Figure Colour changes in anthocyanin dye With according to pH, Ionic properties of EDTA solutions were 1: statistically significant (P<0.001). the Ionode irrigation step using for 2 minutes, the canals were a seriesallofmeasured buffers withpH pHdifferences values from between 8 to 13. The upper Whenwith thepaper pH ofpoints, 5%, 10%, 20% and 40% in solutions electrode, materials dried and the roots placed saline atof using panel (A) shows the visual appearance of a solution of calcium hydroxidefor in 24 water or PEG400 wasany measured every were statistically significant (P<0.001), except for Pulpdent room temperature hours to neutralise remnants of anthocyanin dye as pH alters. Each vial has the same 5 minutes for 2 hours using both a conventional electrode concentration versus Calasept In contrast, with the Solvotrode sodium hypochlorite. of dye.Plus.

The lower panel (B) shows the digital pixel with KCl electrolyte and anof electrode designed electrode, all green To stain the roots, (Ionode), a 3% solution anthocyanin (Red differences and other data for red and channelsbetween for the pHCalmix standards.

There is the pH rises. amaterials consistentwere increase in both parameters specifically alkaline non- aqueous (Solvotrode) Cabbage JiffyforJuice™ powder) in water solvents was prepared. statistically significant as (P<0.001), but there The containing TEABr in removed ethylene from glycol, a solvent was no significant difference between the other three pastes. prepared roots were thePEG saline400 bathasand dried for calcium hydroxide a higher Differences in the readings between the two electrodes were with paper towels andgave paper points, pH andthan the water. canalsWater then coronal andwith outerthesurface of each root, the apex being was sealed solutionswith gavedye pHsolution values which a plateau at 12.7. consistent, Solvotrode electrode always higher irrigated beforereached submerging the roots into molten wax, and Differences each root inthen placed into an Fordye solutions calcium PEG 400, pH with by 0.05-0.16 pH units. the readings between the for 48ofhours.

This hydroxide achieved ainuniform purplethestain which was filledsignificant with anthocyanin increased the withroot. the concentration of calcium hydroxide, and individual the two Eppendorf electrodestube were statistically for all throughout The stained roots were dried with paper solution(P<0.04) to a levelother just below coronal surface of the there and wasmounted a significant positive association with dye materials towels upright using sticky wax onto a holder than fortheOdontocide, where the tooth.

The the samples then maintained at 37˚C in an concentration (P<0.001) (Tableof 3).

Ionode so that the coronal surface the With root the could trend was same were (P=0.6065). be for 3 weeks. electrode, the using pH differential betweenunder 5% and 40% mixtures incubator The measured pH values for Odontocide, which contains photographed a digital camera fixed conditions Digital photographs of the surface of the were was 1.87, and distance 1.74 with the Solvotrode 20% calcium hydroxide in coronal PEG 400 as well asroots ibuprofen, (constant lighting, and exposure settings)electrode. together 1, 2, 3, 5, different 7, 14, and days.

Differences the readings the two electrodes were taken were after not significantly from21 those of Image the 5%analysis calcium with a colourinreference card between and a calibration ruler, to obtain undertaken CS6 each was hydroxide in PEGusing 400Adobe mixture.Photoshop™ In contrast, the pHExtended values for aconsistent, baseline and imagewere as astatistically reference significant point fromat which software to track changes in each roothydroxide over time,in and concentration (P<0.02).

Thebe Solvotrode electrode recorded Calmix, which contains 37.5% calcium PEG subsequent changes could measured.

The roots were the significantly colour change in red and green channels higher pHrandomly values byassigned 0.19-0.36 units forofall3 mixtures.

The calculate 400, were higher than those assigned to pH 5 groups each. One obtained for from 40% the baseline using in a PEG repeated non-aqueous electrodecontrol consistently significantly served as an untreated in whichrecorded no medicament was calcium hydroxide 400 measures (P<0.05). assessment. The selected area of interest was located 500 microns from the higher pH 0.19-0.36 pH units all mixtures placed.

Thevalues four by treatment groups wereforPEG 400 asand a canal walls, as this into was roots considered the greatest depth medicaments pH increased by in 1.74-1.87 pH root vehicle control,(P<0.02).

20% calcium hydroxide PEG 400, Hydroxyl ion release into the tubules that microorganisms would be found. units between theCalmix.

5% andThe 40%materials calcium were hydroxide mixtures Odontocide and injected into Tracking the extent of colour change overlikely 3 weeks in the The selected (10 Xthat10there pixels) wasnoidentical in PEG 400 (P<0.001) withpressure the KCl until and excess TEABr was electrodes the root canal under positive seen stained roots area revealed were changesonin sequential images eachcanals root. orPixel information torespectively. extrude apically. After removing excess material on the untreated roots withofopen in canals treated was with 46 48 INTERNATIONAL INTERNATIONALDENTISTRY DENTISTRY––AUSTRALASIAN AUSTRALASIANEDITION EDITION VOL. VOL.11, 11,NO. NO.44

089)

.118)

ch row,

aline pH the two always king of Calmix, ith both materials Ionode materials Pulpdent votrode d other but there e pastes. des were g higher between for all here the

contains uprofen, calcium alues for in PEG for 40%

ks in the nges in ted with

T ETOEHO H / A/ TAHTAHNAANSASSI A S IDAI D S I /S W / A WLAS LHS H

Table Table 3. The 3. The pH of pHcalcium of calcium hydroxide hydroxide in PEG in PEG 400400 Ca(OH) Ca(OH) 2 2 concentration concentration Ionode Ionode electrode electrode Solvotrode Solvotrode electrode electrode

5% 5%

10%10%

20 % 20 %

40%40%

12.918 12.918 d (0.115) d (0.115) 13.344 13.344 c (0.117) c (0.117)

13.938 13.938 b (0.066) b (0.066)

14.75 a (0.089) 14.75 a (0.089)

13.260 13.260 d (0.083) d (0.083) 13.598 13.598 c (0.063) c (0.063)

14.298 b (0.078) 14.298 b (0.078)

14.940 a (0.118) 14.940 a (0.118)

DataData show show the mean the mean andand SD from SD from five five independent independent experiments. experiments.

Letters Letters indicate indicate statistical statistical groupings groupings across across eacheach row,row, ranked ranked fromfrom highest highest pH (a) pH to (a)lowest to lowest pH (d). pH (d).

collected collected usingusing the histogram the histogram tool,tool, which which gavegave red and red and green green Ionic Ionic properties properties of medicaments of medicaments values values for the for selected the selected areaarea on aonscale a scale withwith 256256 points. points.

The The Medicaments Medicaments based based on PEG400 on PEG400 hadhad a more a more alkaline alkaline pH pH extent extent of change of change fromfrom baseline baseline waswas calculated calculated for the for three the three (Table (Table 4). 4).

Differences Differences in the in the readings readings between between the the two two replicates replicates for each for each group, group, andand analysed analysed using using GraphPad GraphPad electrodes electrodes werewere consistent, consistent, withwith the TEABr the TEABr electrode electrode always always Prism™ Prism™ software software applying applying non-parametric non-parametric analyses analyses for for being being higher higher by by 0.05-0.16 0.05-0.16 pH pH units. units.

The The ranking ranking of of repeated repeated samples samples as well as well as intra-group comparisons. as intra-group comparisons. medicaments medicaments fromfrom mostmost alkaline to least alkaline waswas Calmix, alkaline to least alkaline Calmix, Odontocide, Pulpdent andand thenthen Calasept Plus,Plus, Odontocide, Pulpdent Calasept withwith bothboth Results Results electrodes.

Measured differences between materials electrodes.

Measured pH pH differences between materials Ionic Ionic properties properties of solutions of solutions statistically significant (P<0.001).

Ionode werewere statistically significant (P<0.001).

WithWith the the Ionode When When the the pH pH of 5%, of 5%, 10%, 10%, 20%20% andand 40%40% solutions solutions of of electrode, electrode, all measured differences between materials all measured pH pH differences between materials calcium calcium hydroxide hydroxide in water in water or PEG400 or PEG400 waswas measured measured every every werewere statistically significant (P<0.001), except for Pulpdent statistically significant (P<0.001), except for Pulpdent 5 minutes 5 minutes for 2forhours 2 hours using using bothboth a conventional a conventional electrode electrode versus versus Calasept In contrast, Solvotrode Calasept Plus.Plus.

In contrast, withwith the the Solvotrode withwith KCl KCl electrolyte electrolyte (Ionode), (Ionode), andand an electrode an electrode designed designed electrode, electrode, all all differences between Calmix andand otherother differences between Calmix specifically specifically for alkaline for alkaline non-nonaqueous aqueous solvents solvents (Solvotrode) (Solvotrode) materials werewere statistically significant (P<0.001), but but materials statistically significant (P<0.001), therethere containing containing TEABr TEABr in ethylene in ethylene glycol, glycol, PEGPEG 400400 as aassolvent a solvent waswas no significant difference between the other pastes. no significant difference between the other threethree pastes. for calcium hydroxide gavegave a higher pH than water.

Water for calcium hydroxide a higher pH than water.

Water Differences Differences in readings the readings between the two electrodes in the between the two electrodes werewere solutions gavegave pH values which reached solutions pH values which reached a plateau at 12.7. consistent, a plateau at 12.7. consistent, the Solvotrode electrode always being higher withwith the Solvotrode electrode always being higher solutions of calcium hydroxide in PEG 400, For For solutions of calcium hydroxide in PEG 400, the the pH pH by 0.05-0.16 by 0.05-0.16 pH units.

Differences in readings the readings between pH units.

Differences in the between increased the concentration of calcium hydroxide, increased withwith the concentration of calcium hydroxide, andand the the electrodes statistically significant two two electrodes werewere statistically significant for for all all a significant positive association therethere waswas a significant positive association withwith materials materials (P<0.04) otherother thanthan for Odontocide, where the the (P<0.04) for Odontocide, where concentration (P<0.001) (Table Ionode trendtrend concentration (P<0.001) (Table 3). 3).

WithWith the the Ionode waswas the same (P=0.6065). the same (P=0.6065). electrode, the differential pH differential between 5% and mixtures The The electrode, the pH between 5% and 40%40% mixtures measured measured pH values for Odontocide, which contains pH values for Odontocide, which contains 1.87, 1.74 waswas 1.87, andand 1.74 withwith the the Solvotrode electrode.

Solvotrode electrode. 20%20% calcium hydroxide in PEG as well as ibuprofen, calcium hydroxide in PEG 400400 as well as ibuprofen, Differences in the between the two electrodes werewere werewere Differences in readings the readings between the two electrodes not significantly different of 5% the calcium 5% calcium not significantly different fromfrom thosethose of the consistent, consistent, statistically significant at each hydroxide andand werewere statistically significant at each hydroxide in PEG mixture.

In contrast, the values pH values in PEG 400400 mixture.

In contrast, the pH for for concentration (P<0.02).

Solvotrode electrode recorded Calmix, concentration (P<0.02).

The The Solvotrode electrode recorded Calmix, which contains 37.5% calcium hydroxide in PEG which contains 37.5% calcium hydroxide in PEG higher pH values by 0.19-0.36 pH units formixtures. all mixtures. higher pH values by 0.19-0.36 pH units for all The The 400, 400, significantly higher werewere significantly higher thanthan thosethose obtained for 40% obtained for 40% non-aqueous electrode consistently recorded significantly calcium non-aqueous electrode consistently recorded significantly hydroxide in PEG 400400 (P<0.05). calcium hydroxide in PEG (P<0.05). higher pH values by 0.19-0.36 pH units formixtures all mixtures higher pH values by 0.19-0.36 pH units for all andand medicaments (P<0.02). medicaments (P<0.02). pH pH increased by 1.74-1.87 pH pH Hydroxyl increased by 1.74-1.87 Hydroxyl ion ion release release intointo roots roots unitsunits between the 5% andand 40%40% calcium hydroxide mixtures between the 5% calcium hydroxide mixtures Tracking Tracking the extent the extent of colour of colour change change overover 3 weeks 3 weeks in the in the in PEG 400400 (P<0.001) in PEG (P<0.001) the KCl TEABr electrodes stained withwith the KCl andand TEABr electrodes stained rootsroots revealed revealed that that therethere werewere no changes no changes in in respectively. respectively. untreated untreated rootsroots withwith open open canals canals or inorcanals in canals treated treated withwith 48 48 INTERNATIONAL INTERNATIONAL DENTISTRY DENTISTRY – AUSTRALASIAN – AUSTRALASIAN EDITION EDITION VOL.VOL. 11, NO. 11, NO. 4 4

T E O H / A T H A N A S S I A D I S / W ATLESOHH / A T H A N A S S I A D I S / W A L S H

PEG 400 only (Fig. 2). There were obvious changes in the stained roots over the 3 week period of the experiment, with the characteristic change from a deep purple-blue baseline colour to a yellow colour for the dentine adjacent to the root canal. From gross observation, such changes were most apparent with Calmix (Fig. 3). Quantitative analysis of changes in colour revealed positive changes in both green and red pixel data at 500 microns from the canal wall with each of the three calcium hydroxide materials, indicating that elevations in the pH of the root dentine had occurred progressively over the first 7 days and had then begun to stabilize. The groups were ranked in terms of greatest change to least as Calmix > Odontocide > 20% calcium hydroxide in PEG. Differences between treatments were statistically significant at 7 days and beyond for green and red channel data, with Calmix superior to other groups. After 7 days, the colour change continued to rise slowly for Calmix but began to decline for Odontocide, indicative of buffering of hydroxyl ions by dentine.

Discussion The results of this study provide several insights into the behaviour of calcium hydroxide in different solvents. The results of the first part of the study showed that compared to water, polyethylene glycol 400 when used as a solvent allows high pH values to be achieved, which are above

pH 12.4, the nominal limit for aqueous pastes. While PEG 400 was first used as a viscosity modifier in the endodontic paste Calen in the 1970’s, its ability to alter hydroxyl ion release is a novel finding. The present results indicate that PEG 400 is a suitable solvent for calcium hydroxide.

Chemically, this is due to the large number of ethylene oxide groups along its backbone, which allows PEG to form complexes with metal cations, including calcium ions.7 Such binding of calcium ions drives the dissociation of calcium hydroxide, thus releasing more free hydroxyl ions. This notion is supported by the quantitative results of the root dentine experiment, which showed greater levels of available ions at the same location in the dentine.

PEG 400 is a colourless water soluble and hygroscopic polymer that is miscible with water in all proportions, which explains why PEG 400 can be used as a solvent in its own right with or without the addition of water. PEG is classified as “Generally Recognized as Safe” and has high biocompatibility.7 It has a suitable viscosity for delivery through a fine tip. When used without any added water, as in the case of Calmix, the material will not dry out over time.

Calcium hydroxide pastes produce a high alkaline pH when measured directly with pH electrodes, as shown in the second part of this study, but under clinical conditions the released hydroxyl ions have to diffuse throughout the dentine, during which some buffering will occur from both inorganic and organic components. A saturated aqueous solution of

Table 4. The pH of commercial endodontic pastes measured using two different electrodes

wing ments n and ntine.

Figure 2: Quantitative assessment of changes from baseline in pixel Figure 3: Typical examples of anthocyanin-stained roots showing data for the red (upper) and green (lower) channels for a sample to hydroxyl ion release from medicaments Pulpdent Calaseptcolour Plus changes dueOdontocide Calmix area located 500 microns from the canal wall in the radicular over time, with the colour shifting from purple-blue to green and dentine. The effects of two pastes containing PEG 400 (Calmix in then yellow as pH rises from the canal walls through the dentine.

Ionode 12.706 c 12.662 c 13.170 b 14.996 a green and Odontocide in red) are compared to 20% calcium electrode (0.006) (0.017) (0.025) (0.010) hydroxide in PEG (grey). PEG 400 alone (purple) and an empty canal (black). Data are the mean of three independent experiments.

stroy ll as oxide cient ntine ained ntine, ularly h are nt. E.

(0.008) (0.012)on effective release (0.450) of hydroxide ions,(0.108) which destroy calciumelectrode hydroxide is easily buffered by dentine8 as has been phospholipids of microbial cell membranes as well as seen when dentine powder is added to saturated calcium 9,10 hydroxide solutions. This emphasizes . It is therefore important that hydroxide pH value > 12.0 the rationale behind 12.4 bacterial DNA 12.5 >13.5 third part of the study, which measured alkalinizing effects in ions penetrate into the dentine of the root in sufficient stated by radicular dentine using anthocynanin dye. This dye was concentration to overwhelm the buffering effects of dentine chosenmanufacturer because colour changes correspond to pH changes and cause a highly alkaline pH environment to be sustained both within the root canal system and the adjacent dentine, in a predictable manner giving a gradual change between Data show mean andto SD from fivethen independent experiments.

Letters across each row, in order to indicate suppressstatistical growth ofgroupings microorganisms, particularly colours, fromthe purple-blue green and to yellow as pH ranked from pH alkaline (a) to lowest stated such by the obtained from resistantpH species as manufacturer Enterococcus was faecalis, which are increases intohighest the highly range.pH (c). Information on product material safety data activity sheets and manufacturer websites. able to survive and form biofilms in a pH 10 environment. E. The antimicrobial of calcium hydroxide is based

Solvotrode

12.862 b

12.710 b

13.062 b

15.162 a

VOL. 11, NO. 4 INTERNATIONAL DENTISTRY – AUSTRALASIAN EDITION 49 50 INTERNATIONAL DENTISTRY – AUSTRALASIAN EDITION VOL. 11, NO. 4

T ETOEHO H / A/ TAHTAHNAANSASSI A S IDAI D S I /S W / A WLAS LHS H

showing caments een and dentine.

Figure Figure 2: Quantitative 2: Quantitative assessment assessment of changes of changes fromfrom baseline baseline in pixel in pixel Figure Figure 3: Typical 3: Typical examples examples of anthocyanin-stained of anthocyanin-stained rootsroots showing showing datadata for the for red the (upper) red (upper) and and green green (lower) (lower) channels channels for afor sample a sample colour colour changes changes due due to hydroxyl to hydroxyl ion release ion release fromfrom medicaments medicaments areaarea located located 500500 microns microns fromfrom the canal the canal wall wall in the in radicular the radicular overover time,time, withwith the colour the colour shifting shifting fromfrom purple-blue purple-blue to green to green and and dentine. dentine.

The effects The effects of two of pastes two pastes containing containing PEGPEG 400400 (Calmix (Calmix in in thenthen yellow yellow as pH asrises pH rises fromfrom the canal the canal wallswalls through through the dentine. the dentine. green green and and Odontocide Odontocide in red) in red) are are compared compared to 20% to 20% calcium calcium hydroxide hydroxide in PEG in PEG (grey). (grey).

PEGPEG 400400 alone alone (purple) (purple) and and an empty an empty of three of three independent independent experiments. experiments. canalcanal (black). (black).

DataData are the aremean the mean

destroy well as droxide ufficient dentine ustained dentine, rticularly hich are ment. E.

8 effective release of hydroxide which destroy effective release of hydroxide ions,ions, which destroy calcium calcium hydroxide hydroxide is easily is easily buffered buffered by dentine by dentine as 8has as has beenbeen on on phospholipids of microbial membranes as well of microbial cell cell membranes as well as as seenseen when when dentine dentine powder powder is added is added to saturated to saturated calcium calcium phospholipids 9,10 9,10 bacterial DNA DNA hydroxide hydroxide solutions. solutions.

This This emphasizes emphasizes the rationale the rationale behind behind bacterial . It is. Ittherefore is therefore important important that that hydroxide hydroxide thirdthird partpart of the of study, the study, which which measured measured alkalinizing alkalinizing effects effects in in ionsions penetrate penetrate into into the the dentine dentine of the of the rootroot in sufficient in sufficient radicular radicular dentine dentine using using anthocynanin anthocynanin dye.dye.

This This dye dye waswas concentration concentration to overwhelm to overwhelm the buffering effects of dentine the buffering effects of dentine chosen chosen because because colour colour changes correspond to pH cause a highly alkaline pH environment be sustained cause a highly alkaline pH environment to betosustained changes correspond to changes pH changes andand in ainpredictable manner giving a gradual change between within the root canal system the adjacent dentine, within the root canal system andand the adjacent dentine, a predictable manner giving a gradual change between bothboth colours, in order to suppress growth of microorganisms, particularly to suppress growth of microorganisms, particularly colours, purple-blue to green to yellow as pH in order fromfrom purple-blue to green andand thenthen to yellow as pH resistant species as Enterococcus faecalis, which resistant species suchsuch as Enterococcus faecalis, which are are increases the highly alkaline range. increases into into the highly alkaline range.

10 to survive biofilms a 10 pHenvironment. to survive andand formform biofilms in a inpH antimicrobial activity of calcium hydroxide is based ableable The The antimicrobial activity of calcium hydroxide is based environment.

E. E.

50 50 INTERNATIONAL INTERNATIONAL DENTISTRY DENTISTRY â&#x20AC;&#x201C; AUSTRALASIAN â&#x20AC;&#x201C; AUSTRALASIAN EDITION EDITION VOL.VOL. 11, NO. 11, NO. 4 4

droxide de as as reau Stds Stds

lete drug drug cal ress,Press,

ents. solvents.

nonand nonation, h titration, nitor bath bath -24. gers RD. RD. olglycol as as 82. alsh J. AnLJ. An ndodontic ontic 2985(3):298-

ase: free free 12-26.

6. nscriptome tome oaline alkaline

aline . Alkaline crosson, crosspl viron Environ

rigdor D. D. occus erococcus 35:221-8.

21-8. slvents Part Part ssociation ation data. etric data.

T ETOEHO H / A/ TAHTAHNAANSASSI A S IDAI D S I /S W / A WLAS LHS H

Suppl):S64-82. faecalis faecalis is however is however inactivated inactivated by alkaline by alkaline environments environments at at Suppl):S64-82.

11,1211,12 3. Bates Bower Smith ER. Calcium hydroxide pH values pH values around around 11.5 11.5 to 11.9 to 11.9 RG,RG, Bower VE, VE, Smith ER. Calcium hydroxide as as . The. The rationale rationale for using for using 3. Bates a highly alkaline standard.

J Res Bureau alkaline pH pH standard.

J Res NatNat Bureau StdsStds a non-water a non-water solvent solvent suchsuch as PEG as PEG 400400 is toishelp to help achieve achieve a a a highly 1956;56(6):305-12. sufficiently sufficiently highhigh pH pH at the at the canal canal wallswalls andand into into the the innerinner 1956;56(6):305-12.

4. Sweetman S (ed).

Martindale: complete 4. Sweetman S (ed).

Martindale: the the complete drugdrug radicular radicular dentine dentine to reach to reach this objective. this objective. reference. edition.

London: Pharmaceutical Press, reference.

36th36th edition.

London: Pharmaceutical Press, 2009: p. 2272.

2009: p. 2272.

Conclusions Conclusions 5. Tremillon Chemistry in non-aqueous solvents.

B. B. Chemistry in non-aqueous solvents.

While While the the concepts concepts of ionic of ionic equilibria equilibria andand pH pH in nonin non- 5. Tremillon Netherlands: Springer, Springer, 1974. aqueous aqueous solvents solvents havehave beenbeen wellwell documented documented in the in literature the literature Netherlands: 1974.

13 13 J. pH Measurement in aqueous andand non-nonfor many years 6. Quitmeyer J. pH Measurement in aqueous for many years , the, chemical the chemical properties properties of such of such materials materials 6. Quitmeyer solutions: When usedused in combination aqueous solutions: When in combination titration, as vehicles as vehicles for calcium for calcium hydroxide hydroxide in dentistry in dentistry has has not hitherto not hitherto aqueous withwith titration, measurement a simple method to monitor beenbeen explored. explored.

This This studystudy has has shown shown that that PEGPEG 400400 is a is a pH pH measurement is ais simple method to monitor bathbath concentration.

Metal Finishing 2008:106(10):21-24. suitable suitable vehicle vehicle for calcium for calcium hydroxide, hydroxide, andand givesgives higher higher pH pH concentration.

Metal Finishing 2008:106(10):21-24.

7. Chen J, Spear Huddleston Rogers values values thanthan can can be be achieved achieved in aqueous in aqueous solutions. solutions. 7. Chen J, Spear SK, SK, Huddleston JG, JG, Rogers RD. RD. Polyethylene glycol solutions of polyethylene glycol Commercial Commercial medicaments medicaments which which use use PEGPEG without water glycol andand solutions of polyethylene glycol as as without water Polyethylene green reaction media.

Green Chem 2005;&:64-82. show higher measured pH pH andand greater pH pH change in in green reaction media.

Green Chem 2005;&:64-82. show higher measured greater change 8. Athanassiadis B, Abbott PV, George radicular dentine thanthan B, Abbott PV, George N, Walsh LJ. An radicular dentine which contain mixed N, Walsh LJ. An thosethose which contain PEGPEG mixed withwith 8. Athanassiadis studystudy of the by dentine of some endodontic water, or water alone. results of present the present of inactivation the inactivation by dentine of some endodontic water, or water alone.

The The results of the studystudy alsoalso vitrovitro and and show potential of anthocyanin staining a method medicaments medicaments bases.

J. 2010;55(3):298show the the potential of anthocyanin staining as aasmethod theirtheir bases.

AustAust DentDent J. 2010;55(3):298which show fluctuations in root dentine caused 305. which can can show pH pH fluctuations in root dentine caused by by 305. endodontic medicaments. experimental approaches 9. Freeman 9. Freeman Crapo Biology of disease: endodontic medicaments.

SuchSuch experimental approaches BA, BA, Crapo JD. JD. Biology of disease: free free be useful to inform the development of products radicals tissue injury.

Invest 1982; 47:412-26. maymay be useful to inform the development of products which andand tissue injury.

Lab Lab Invest 1982; 47:412-26. which radicals S, B, LiuJiang B, Jiang Z, Liang J. Transcriptome can can achieve the desired pH changes in the dentine for for 10. 10.

Ran Ran S, Liu W, W, Sun Sun Z, Liang J. Transcriptome achieve the desired pH changes in inner the inner dentine analysis of Enterococcus faecalis in response to alkaline effective disinfection. analysis of Enterococcus faecalis in response to alkaline effective disinfection. stress.

Microbiol.

2015;6 (795):1-11. stress.

FrontFront Microbiol.

2015;6 (795):1-11.

11. 11.

Flahaut S, Hartke A, Giard JC, JC, Auffray Y. Alkaline Flahaut S, Hartke A, Giard Auffray Y. Alkaline Conflict Conflict of interest of interest statement statement response in Enterococcus faecalis: stress response in Enterococcus faecalis: adaptation, crossadaptation, crossTwoTwo of the of the authors authors (BA (BA andand LW)LW) are are co-inventors co-inventors of of stress protection, changes in protein synthesis.

Environ protection, andand changes in protein synthesis.

ApplAppl Environ Calmix Calmix paste. paste.

Microbiol 1997;63:812-4.

Microbiol 1997;63:812-4.

Evans Davies Sundqvist Figdor 12. 12.

Evans M, M, Davies JK, JK, Sundqvist G, G, Figdor D. D. References References Mechanisms involved in the resistance of Enterococcus involved in the resistance of Enterococcus 1. Fava 1. Fava LR, Saunders LR, Saunders WP.WP. Calcium Calcium hydroxide hydroxide pastes: pastes: Mechanisms faecalis to calcium hydroxide.

Int Endod J 2002; 35:221-8. to calcium hydroxide.

Int Endod J 2002; 35:221-8. classification classification andand clinical clinical indications. indications.

Int Int Endod Endod J. J. faecalis Roses M. Ionic equilibria in non-aqueous solvents 13. 13.

Roses M. Ionic equilibria in non-aqueous solvents Part Part 1999;32(4):257-82.

1999;32(4):257-82. equations for calculation of pH, dissociation 2. Athanassiadis 2. Athanassiadis B, Abbott B, Abbott PV, PV, Walsh Walsh LJ. The LJ. The use use of of 1. General 1. General equations for calculation of pH, dissociation andand reference potentials calcium calcium hydroxide, hydroxide, antibiotics antibiotics andand biocides as antimicrobial constants reference potentials potentiometric data. fromfrom potentiometric data. biocides as antimicrobial constants medicaments in endodontics.

AustAust DentDent J. 2007;52(1 Chim 1993;276:211-21.

Chim ActaActa 1993;276:211-21. medicaments in endodontics.

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52 52 INTERNATIONAL INTERNATIONAL DENTISTRY DENTISTRY â&#x20AC;&#x201C; AUSTRALASIAN â&#x20AC;&#x201C; AUSTRALASIAN EDITION EDITION VOL.VOL. 11, NO. 11, NO. 4 4