THE HEMOLYSIS AND CYTOTOXICITY OF A ZEOLITE-CONTAINING ROOT FILLING MATERIAL IN VITRO

David Charles Thom

A thesis submitted in conformity with the requirements for the degm of Master of Science

Graduate Department of Faculty of Dentistry University of Toronto

@Copyright by David Charles Thom 2001

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The Hemolysis and Cytotoxicity of a Zeolite-Containing Root Füling Material In Yiiro Master of Science (Endodontics) 2001 David Charles Thom Faculty of Dentistry, University of Toronto

ABSTRACT

ZUT is an endodontic sealer consisting of glass ionomer cernent (GIC) and an

antimicrobial-containing zeolite (0.2% ~eomic" w/w). Cytotoxicit y and hemol ysis of

ZUT, Ketac-cerna (ZUTs GIC component), Ketacm-~ndo (a GIC), and AH-26'B (a resin;

with/without silver formulations) were characterized. Ce11 death was assessed using a

Milli pore Filter test with a HeLa ce11 monolayer (n= 1 Wsealer material; repeated twice).

Cytotoxicity of materials at various time points (fiesh mixed, 1, 2, 3, 6, and 24 hr. from

mixing) was compared to wax controls. GICs and AH-26@ formulations were non-

cpotoxic 1 and 6 hours afier setting, respectively (1-way ANOVA with Duncan's pairwise

t-tests; p>0.05). Disks of AH 2 6 (with silver) were significantly more hemolytic than the

other test materials after incubation in a saline-rabbit blood solution (n=d/material;

repeated twice) (ANOVA; p<0.00 1 ). ~eomic" did not increase the cytotoxic and

hemolytic potential of Ketac-cema. The degree of cytotoxicity and hemolysis for the ZUT

material was comparable to that of the clinically-accepted ~ e t a c @ - ~ n d o and AH-26"

sealers.

ACKNOWLEDGEMENTS

The contributions of George Adams, John E. Davies, Dentsply Ltd., ESPE Dental

AG., Shimon Friedman, Cheung Lo, Elaine Parker, Stuart Rae, Berthold Reusch, Amy

Shiga and Calvin D. Torneck are gratefully acknowledged. Special thanks to Gajanan

Kulkarni for his statistical support. Funding was provided by an Endowment Fund

Research Grant fiom the Canadian Academy of Endodontics.

This work would not have been possible without the unwavering dedication and

support of J. Paul Santerre. Thank you.

TABLE OF CONTENTS

Abstract

Acknowledgements

Table of Contents

List of Figures

List of Tables

Page

. . 11

... 111

i v

vi i

... Vll l

INTRODUCTION .................................................................................................. I

LITERATURE REVIEW ...................................................................................... 2 Rationale of endodontic treatment ............................................................................ 2 Gutta-percha for endodontic application ................................................................... 4

The shortcomings of gutta-percha as a root filling material .............................. 6 @D AH 26 sealer ........................................................................................................... 9

@ Formaldehyde and AH 26 cytotoxicity ................................................... 11 Glass ionomer cement ........................................................................................... 15

Glass ionomer cement chemistry ................................................................... 16 Biocompatibility of glass ionomer cements ............................................... 18 Glass ionomer cement bonding to dentin .................................................... 19 Resistance to root fkacture ............................................................................. 22 Antibacterial properties of glass ionomer cement ........................................... 26 Clinical studies using glass ionomer cement as a root filling material ........... - 2 8

Zeolites in dentistry ................................................................................................ 29 .............................................................. General structure and characteristics 29

@ .............................................................. Zeomic : a silver-containing zeolite 31 Zeolite incorporation into dental materials ................................................. 34

ZUT: a zeolite-containing glass ionomer cement endodontic sealer ........................ 36

RESEARCH RATIONALE ..................... .. ...................................................... 41 ................................................................................. 3.1) Statement of the problem 41

....................................................................................................... 3.2) Objectives 42 ...................................................................................................... 3.3) Hypothesis 42

......................................................................... METHODS AND MATERIALS 43 ............................................................... 4.1) Rationale and operational definitions 43

.................................................................................................. Assumptions 43 .................................................................................................... Limitations 46

Delimitations ................................................................................................. 47 ................................................................................ 4.2) Selection of test materials 48

4.3) Experimental methods .............................................................................................. 49 4.3.1 ) Percent Hemolysis Test ....................................................................... 49

Materials ...................................................................................... 50 ............................................................ Preparation of rabbit blood 51 ........................................................... Preparation of test materials 51

Met hodology ................................................................................. 52 4.3 -2) The necessity of preliminary testing ............................ ... ............... 53 4.3 -3) Modifications to the Percent Hemolysis Test protocol ......................... 53

The effect of test matenal surface area ........................................... 53 ............................... Potassium oxalate as an anticoagulation agent 54

The importance of the blood-saline vo1ume:test material ............................................................................... mass ratio 55

.................... Determination of inherent error in this testing system 56 Possible effect of soluble products on spectrophotometric

readings .................................................................................. 57 4.3 -4) MiIlipore Filter Test of cytotoxicity ..................................................... 58

Materials ........................... .., ...................................................... 59 Culturing of the HeLa ce11 line ................................................. 61 Determination of ce11 concentration ............................................... 63 Preparation of the agar medium ..................................................... 64

............................... Preparation of the millipore filter ce11 cultures 64 Preparation of test materials .............. .. ........................................ 65

................................................................................. Methodology 65 ...................................................... Assessment of millipore filters 66

.......................... ........... Interpretation of millipore filter scores ... 66 .............................. 4.3.5) Modifications to the Millipore Filter Test protocol 67

.............. Effect of HeLa ce11 confluence on filter staining intensity 67 ................................................................................................... 4.4) Data analysis 68

....................................................................... 4.4.1) Percent Hemolysis Test 69 ...................................... .................... ........ 4.4.2) Millipore Filter Test .. .... 69

............................................................................. 4.5) Resources and environment 69

................................................................................................................. 5) RESULTS 70 5.1) Percent Hemolysis Test results using cylinders of matenals ............................. 70 5.2) Percent Hemolysis Tests using disks of material .............................................. 71 5.3) Results of the Millipore Filter Test of cytotoxity ............................................. 73

........................................................................................................... 6) DISCUSSION 76 ...................................................................... ZUT as a potential endodontic sealer 76

Hemolytic properties of the test materials ............................................................... 77 ............................................................................ Cytotoxicity of the test materials 80

.............................................................................................. Future investigations 84

...................................................................................................... 7) CONCLUSIONS 86

LIST OF FIGURES

Page

FIG-GRE 1 . FIGURE 2 . FIGURE 3 . FIGURE 4 . FIGURE 5 . FIGURE 6 . FIGURE 7 . FIGURE 8 .

Endociontical1 y-treated tooth with exposed gutta-percha ................... .. ..... 7

Illustration of a hexane molecule contained within a zeolite structure ....... 30

QD .................................. Scanning electron micrograph of Zeomic particles 33

Sumrnary of the Percent Hemolysis Test methodology .............................. 57

.......... Summary of the Millipore Filter Test of Cytotoxicity methodology 68

................ Percent Hemolysis Test results using cylinders of test materials 70

Percent Hemolysis Test results using disks of test materials ...................... 71

.............................................. Millipore Filter Test of Cytotoxicity results 75

LIST OF TABLES

Page

TABLE 1 . TABLE 2 . TABLE 3 . TABLE 4 . TABLE 5 . TABLE 6 . TABLE 7 . TABLE 8 .

TABLE 9 . TABLE 10 . TABLE 11 . TABLE 12.

TABLE 13 . TABLE 14 .

TABLE 15 . TABLE 16 .

Requirements for an ideal root canal filling material ................................... 3

Constituent percent weights of gutta-percha endodontic filling materials .... 5

Composition of AH 26@ (with silver) endodontic sealer ............................ 10

Composition of AH 26@ (silver-free) endodontic sealer ............................ I 0

aD Composition of AH Plus endodontic sealer ............................................. 10

Physical and chemical properties of formaldehyde .................................... 11

........................................ Sources of formaldehyde production and intake 12

Formaldehyde detected in AH 26@ and ~ 2 " samples ............................................................................ at different setting times 14

fa ..................... ............................ The compostition of Ketac-Cem ...... 17

@ ................................................. The composition of Ketac -Endo Aplicap 18

Force to fiacture in roots obturated with different materials ...................... 24

................................ Resistance to fixture of endodontically treated roots 25

Physical and chemical characteristics of ~eomic@ ..................................... 34

Apical leakage in vifro of three obturation techniques using three ................................................................................ evaluation techniques 39

Cell lines used to evaluate the cytotoxicity of endodontic materials .......... 44

Optical density (OD) readings of test materials ......................................... 72

viii

1) INTRODUCTlON

Glass ionomer cement (GIC) has been introduced as a possible root filling material

(Pitt Ford, 1979) and shown to possess potentially superior sealing (Koch et al., 1994) and

antimicrobial properties over conventional sealers. This material is able to chemically and

mechanically bind to dentin, enhancing the seal and reinforcing the tooth (Ray and Seltzer,

199 1 ; Saunders et al., 1992). Although the antimicrobial activity of GIC materials occurs

through its fluoride-releasing action, this effect is variable and diminishes within 24 hours

fiom mixing (Shalhav et al., 1997), rendering the root canal system and its associated

tissues vulnerable to microbial reinfection. An experimental endodontic sealer, ZUT,

consisting of a GIC sealer and an antimicrobial-containing zeolite, has been developed at

the University of Toronto, Faculty of Dentistry. It is intended to provide a prolonged

antimicrobial activity while retaining the sealing, tooth-reinforcing and biocompatible

characteristics of conventional GIC sealers (Patel et al., 2001). Antibacterial (Patel et al.,

2001) and biomechanical investigations (Lalh et ai., 1999) on ZUT have yielded favorable

results but to date no biological testing has been conducted.

2) LITERATURE REVIEW

Rationale of endodontic treatment

Endodontic therapy includes, but is not limited to, the prevention and treatment of

diseases and injuries of the dental pulp and associated periradicular tissues (American

Association of Endodontists, 1998). Kakehashi and associates (1 965) conclusively showed

that the deteminant factor in penradicular tissue healing is the absence of a microbial

flora. Funhermore, the presence of microorganisms in the root canal system after

treatment has been identified as the paramount cause of persistent disease (Sjogren et al.,

1997). The main mechanisms for root canal infection afier treatment are the resistance of

some microorganisms to conventional treatment (Dahlén et al., 2000; Molander et al.,

1998; Nair et al., 1990) and the ingress of a new microbial tlora into the canal space

subsequent to treatment (e.g. through coronal leakage) (Friedman et al., 1997; Torabinejad

et al., 1990).

When disinfection and shaping procedures are completed, the obturation phase of

endodontic treatment is performed, according to Grossman (1988), with the objective of

replacing the dental pulp with an inert hermetic sealing agent. The ideal root filling

materiai should f i l f i l eleven requirements (listed in Table 1) to prevent postoperative

reinfection of the root canal system. Despite satisQing only some of these recommended

requirements, gutta-percha in association with a root canal sealer is presently the most

commonly used root filling material (in much the same manner as they were when first

introduced by Hill in 1847 (Koch and Thorpe, 1909)).

The premise that the entire canal system of pulpless teeth must be filled as a

requirement for healing was presented in the "Hollow Tube Theory" put forth by Rickert

and Dixon in 193 1. These investigators found that hollow platinum needles embedded in

rabbit connective tissue evoked an inflammatory response that was localized to the open

ends. By contrast, inflammation was not observed around implanted solid platinum rods.

Similady, they reported inflammation in association with sterilized implants of teeth with

unobturated root canal systems but not with teeth in which the root canal was obturated.

Table 1. Requirements for an ideal root canal filling material (adapted from Grossman, 1 988)

Easy to manipulate with ample working time. Dimensionally stable afler placement. Able to seal the canal laterally and apically; conforms to anatomical shape and contour of the canal. Does not irritate the periapical tissues. Impervious to moisture, non-porous. Not affected by tissue fluids and insoluble in tissue fluids; does not corrode or oxidize. Bactenostatic or bacteriocidal. Radiopaque, easy to discem on a radiogaph. Does not discolour the tooth structure. Sterile or easily and quickly stenlized prior to insertion. Easily removed from the canal if necessary.

The Hollow Tube Theory was not wholly supported by Tomeck (1966), who

implanted sterilized polyethylene tubes, open at one or both ends, into the subcutaneous

tissues of Wistar rats. Connective tissue ingrowth occurred in open tubes with large

lumens while ingrowth occurred only to a limited extent in single-ended tubes. When

stenle, autoclaved rat muscle was used to fil1 single-ended tubes, a low grade chronic

inflammation was seen immediately adjacent to the muscle. The inflammatory response

was most severe when the muscle was contaminated with endogenous microorganisrns

isolated from the oral cavity (Tomeck, 1967). These findings supponed the Hollow Tube

Theory only if certain conditions prevailed; that is, the presence of microorganisms and

necrotic tissue wit hin the unfi 1 led root canal space. Conversel y, successful healing afler

endodontic treatment can be expected when the canal space is clean and maintained free of

microorganisms, regardless of whether or not the root canal system is entirely obturated.

Obturation of the entire root canal system does play an important role in the overall

treatment objective of promoting periradicular tissue healing. Unlike the straight, smooth

walls o f polyethylene tubing, the works of Hess (1925) and Kuttler (1955) have

demonstrated the multitude of anatornic complexities of the root canal system. It is also a

well-established fact that bacterial species are able to penetrate into the dentinal tubules of

roots associated with periapical lesions (Peters et al., 2001). The many complex

anatomical sites in which microorganisms can reside and fluonsh, emphasize the

importance of Grossman's requirement that the root fiiling material should not only seal

the canal both laterally and apically, but also possess bacteriostatic (or bacteriocidal)

properties to resist bacterial repopulation.

Gutta-percha for endodontic application

Conventional endodontic treatment utilizes both chemical and mechanical means to

debride, disinfect and prepare the root canal space for filling with a material. Gutta-percha

is the most commonly used root filling material. with its use popularized by Bowman in

1867 @owman, 1938). Natural gutta-percha is rubber-like matenal extracted from the sap

of the Taban tree (Isottandra percho). It is a trans-1,4-polyisoprene that is harder, more

brittle, and less elastic than natural rubber, the cis-isomer. Modem endodontic gutta-

percha consists mainly of zinc oxide. About 20% of the endodontic material is synthetic

"'gutta-percha-like material", with heavy metal salts, waxes, and resins making up the

remaining const ituents (Friedman et al.. 1977).

The specific content of the various gutta-percha products is normally kept a

propnetary secret, however Friedman and associates (1977) assayed five commercially

available gutta-percha endodontic products for organic (e.g. gutta-percha, waxes, resins)

and inorganic (e-g. metal sulfates, zinc oxide) components (Table 2).

Table 2. Constituent percentage weights of gutta-percha endodontic filling materials (expressed as mean percentage, standard deviations in brackets) (adapted fiom Friedman et al., 1977)

I Brand I Gutta-percha Wax +/or resin Meta1 sulfates Zinc oxide I I I

~ e r n ~ r ~ t e ' 20.6 (1.4) 2.9 (0.2) 3.4 (2.1) 73.4 (2.0)

%charles B. Schwed Co., New York

~ ~ n o l '

Indian ~ e a d #

' Mynol Chemical Co., Broomall, Pa. 'union Broach Co., Inc., Long Island City, N.Y. Y star Dental Co., Conshohocken, Pa. %stron Corp., Canton, Mass.

Regardless of the brand, the proponion of organic and inorganic fractions were found

to be essentially the same. The mean organic percentage of al1 brands was 23.1% (* 0.5%

standard deviation) while the inorganic percentage was 76.4% (h 0.5% standard deviation).

19.9 (O. 1)

21.8 (0.2)

3.9 (0.2)

1 .O (0.2)

16.2 (1.8)

17.3 (0.3)

59.1 (2.0)

59.6 (0.1)

High levels of gutta-percha produced increased ngidity and strength, while higher zinc

oxide content resulted in increased brittleness and decreased flow .

The zinc oxide component has long been thought to account for the weak

bacteriostatic characteristics show by gutta-percha filling material. Moorer and Genet

(1 982) attempted to identie the bactenostatic component of endodontic gutta-percha cones

using microbiologie analysis, measurement of osmolanty, microscopy, x-ray diffraction

and scanning electron micrograph y. They detennined t hat cry stal line Zn0 (zincite) is

present as a physically bonded (entrapped) material that is leached fiom cones during

contact with water. Upon hydrolysis, the resulting mobile zn2' ions affect the antibacterial

properties exhibited by gutta-percha cones. It was hypothesized that zinc oxide particles

serve as a "depot" or ccreservoir77 for the rapid mobilization of zinc ions that diffise to the

site of any zinc-consuming reaction or possibly to a microorganism receptor site.

The shortcomings of gutta-percha as a root filling material

Although it fulfils some of the desired characteristics of an ideal root filiing

material, gutta-percha does not adhere to the dentin of the root canal walls. As a result, a

sealer material is required to serve as an interface between the solid core filling material

and the root dentin (Cohen and Burns, 1998). Sealer also fills anatomical irregularities that

gutta-percha cones cannot (e.g. dentinal tubules, fins and grooves along the canal wall). A

classical study by Marshall and Massler ( 196 1 ) demonstrated that radioisotope penetration

into root canals filled with gutta-percha and sealer was decreased compared to roots filled

only with gutta-percha. These findings are supported by the more recent study by Wu and

associates (200 l), who found that within 48 hours rwts obturated with gutta-percha and

sealer leak significantly less (p<0.0001) in vitro than mots only filled with gutta-percha.

C haracterist ics of an ideal endodont ic sealer (suggested by Grossman, 1 988) :

Tacky when mixed and has good adhesion to the canal wall. Has ample setting tirne, allowing the clinician to make necessary adjustments to the fiilhg material. Produces a hermetic seal. Powder particles mix easily with the cernent liquid. Radiopaque; able to reveal morphologies such as accessory canals, multiple foramina, resorptive areas, fiacture lines. Expand while setting. Bacteriostatic. Bio logically acceptable, doesn' t irritate periapical tissues. Insoluble in tissue fluids. Does not stain tooth structure. Soluble in common solvents if removal is necessary. Should not provoke an immune response in periapical tissues.* 1s not mutagenic or carcinogenic.*

*suggested in Cohen and Burns ( 1 99 1 )

Despite the overall high success of endodont ic treatment (Sjogren cf al., 1 990),

gutta-percha and sealer are unable to hermeticaily seal the canal against reuifection,

regardless of the technique used for obturation (see Figure 1 ).

Figure 1. Endodonticaüy-treated tooth with exposed gutta-percha root nIlllig. This tooth is at high risk for recontamination of the root canal space.

Khayat and associates (1993) used an in vitro laboratory model to detemine the

time required for bacterial penetration through roots obturated with gutta-percha and

~ 0 t h ' ~ ~ cernent (a zinc oxide and eugenol-based sealer; Roth International Ltd., Chicago,

IL) using samples of human saliva placed in the chambers of the teeth. To determine if the

manner in which roots are obturated has an effect on leakage, both cold lateral and warm

vertical condensation techniques were performed. There were no statistically significant

differences between the two obturation methods. Within 30 days al1 canals were

contaminated.

An in vivo animal model involving bacterial ingress into endodontically treated

teeth was developed by Friedman and associates (1997) to assess the eficacy of root

fillings in the prevention of periradicular inflammation. Roots of beagle dogs were

obturated using gutta-percha and Kerr PCS" (another zinc oxide and eugenol-based sealer;

Kerr Co., Romulus, MI) and allowed to set. The chambers of these teeth were then

inoculated with plaque samples and again restored. Histological examination revealed

signs of mild inflammation occumng in 67% of the roots 14 weeks afier treatment.

Although the tirne periods for bacterial penetration of the canal system determined by in

vzzro and in vivo studies may differ, the ovenvhelming majority of these investigations

conclude that the use of gutta-percha and conventional sealers cannot prevent

recontamination of the root canal system when exposed to microorganisms of the oral

environment.

AH 26@ sealer

This epoxy resin material, developed by Schroeder (1957) has sufficient volume

stability to be used either as a sealer with gutta-percha cones or as a mot fiiling materiai on

its own. The long setting time [9 - 15 hours at 37 OC; ISO 6876:1986(E), Dentsply

DeTrey monogram] and matenal fluidity of AH 26" have been suggested to prevent

cracking and rapid separation from the dent inai wails (De Gee et al., 1994). Silver particles

enhance the radiopacity of the root filling material as well as providing a degree of

antimicrobial activity. Concerns regarding the potential cytotoxicity and visible staining

produced by the silver particles have resulted in a silver-free formulation of AH 26". A

newer version of this sealer is AH plus* (Dentsply DeTrey GmbH, Konstanz, Germany),

which does not contain the catal yt ic amine hexamethy lene tetramine, thought to produce

formaldehyde during the setting reaction of AH 26@ formulations. This new formulation,

however, is reported to have inferior physical characteristics. Laboratory in v i m

investigations by Zmener and associates (1997) suggested AH PIUS@ to allow greater

leakage in obturated teeth than AH 26: possibly because of a faster setting time causing

shrinkage stress and debonding from the canal walls. The compositions of the onginal AH

26" (with silver), AH 26@ silver-free, and AH plusm formulations are listed in Tables 3 - 5 .

Table 3. Composition of AH 26@ (with silver) endodontic sealer (adapted fiom Dentsply DeTrey GmbH monogram)

bismuth (III) oxide (60.0) hexamethylene tetramine (25 .O) titanium dioxide (5.0)

Powder (% weight)

bis-phenol diglycidyl ether (1 00.0) I

Liquid

Table 4. Composition of AH 26@ (silver-fiee) endodontic sealer (adapted fiom Dentsply DeTrey GmbH monogram)

Powder (% weight)

1 Liquid

Table 5. Composition of AH plus@ endodontic sealer (adapted from Leyhausen et al., 1999)

bismuth (III) oxide (80.0) hexamethylene tetramine (20.0)

I

bis-phenol diglycidyl ether (1 00.0)

-- - -

epoxy resin calcium tungstate zirconium oxide iron oxide aerosil

Paste A

adamantaneamine N,N-dibenzyl-5-oxanonane-diamine- 1,9 TCD-diamine calcium tungstate zirconium oxide aerosil silicone oil

Paste B

Formaldehyde and AB 26@ cytotoxicity

Spingberg and associates (1993) utilized gas chromatography and mass

spectroscopy to detemine the presence of fomaldehyde in both AH 26@ (with silver) and

~ 2 " (Indrag Agsa, Switzerland) root canal sealer matenals. Discovered in 1859 and later

used as a disinfectant, formaldehyde (FA) is a flammable and colourless chernical readily

soluble in polar solvents and most commonly available as a 3040% aqueous solution

(Table 6). Today it is a significant chemical commodity consumed worldwide at a rate

exceeding tive billion kilograrns per year (ECIETC Technical Report, 1982).

Table 6. Physical and chemical properties of fomaldehyde (adapted fiom Environmental Health Criteria, 1989)

IUPAC name 1 met hanal

chemical formula CH20 BCHO]

Common synonyms formaldehyde, met hanal, met hy lene oxide, oxymethylene, methylaldehye,

oxomet hane

Comrnon names for solutions of formaldehyde

formalin, formol

Relative molecular mass 30.03

Melting point (OC) -1 18

Vapour pressure 101.3 kPa at -19 OC 52.6 kPa at -33 OC

Specific reaction rate (k) with OH radical (k OH)

15 10"'m3/mol s

Two important sources are automotive engine exhaust and wastes produced during

the manufacture of FA or associated matenals. Formaldehyde intermediate products are

also found in wood products, adhesives, insulation, resins, explosives, lubricants, and

fertilizers. Other common sources of FA exposure are outlined in Table 7.

Table 7. Sources of forrnaldehyde production and intake (adapted fiom Environmental Healt h Criteria, 1989)

Source

Car engine exhaust

Rainwater

Quantity/Concentration of Formaldehyde

700 mg FA& fiel

1 10- 174 pg FAIL water

Drinking water

Mean daily intake fiom drinking water alone

"Conversion Factors: 1 ppm formaldehyde = 1.2 mg/m3 at 25 OC, 1066 mbar 1 mg formaldehyde/m3 = 0.83 ppm

0.1 mg FA/L water

0.2 mg FA

Mean daily intake from food ingested

Pear

Tobacco smoke

Formaldehyde is readily absorbed in both the respiratory and gastrointestinal tracts

where rapid metabolism occurs so that blood concentrations do not increase appreciably.

The FA metabolites are eliminated in the expired air and urine afier their incorporation into

macromolecules (Environmental Healt h Criteria, 1989).

Evidence suggests FA does not have a high carcinogenic potential and although it

interferes with D N A repair in human cells Nt vitro, there are no data relating to mutagenic

outcornes. One conclusion by the International Programme on Chemical Safety (IPCS)

1.4- 14 mg FA

38 - 60 mg FA/kg fniit

48 mg FA.^' air

regarding exposure to FA and endodontic procedures was, in order to avoid adverse

reactions in dental practice, not to extrude root canal sealers beyond the apex in short-term

exposure situations (Environmental Healt h Criteria, 1989).

Koch and associates (2001) studied the release of formaldehyde fiom ground AH

26@ and ~ 2 " samples allowed to set for a period of six months. Using high-performance

liquid chromatography, AH 26@ material had a significantly higher (one-tailed t-test;

p<0.000 1 ) FA release (6.6 * 2.6 pg/mg material) than ~ 2 " (0.3 0.1 pg/mg material).

Spingberg and associates (1993) did not detect FA in either the powder or liquid

resin components of AH 26@, but there was a measurable level in freshly-mixed samples

and also in materials allowed to set for a seven day period (see Table 8). Both AH 2%

sealers contain the catalytic amine hexamethylenetetramine (HMT) synthesized from

formaldehyde and ammonia. They concluded that formaldehyde is the result of HMT

decomposition during the setting reaction of AH 26@. Decomposition can occur in water

solution and acidic environments, similar to those encountered in the periradicular tissues

during endodontic treatment .

These findings correlate with studies suggesting a high in ÿitro cytotoxicity of AH

26@ immediately afier mixing which is reduced to a relatively inert level after setting

(Sphgberg, 1969; Sphgberg and Langeland, 1973). Some investigators have reported a

higher incidence of intestinal tissue dysplasias in rats when exposed to daily doses

exceeding 100 mg fonnaldehydekg body weight/day (Le. 100 ppdday) for periods of 1-2

years (Furihata et ai., 1988). Leyhausen and associates (1999) suggested the cytotoxic

andor mutagenic effects of AH 26@ may also be attributed to epoxy derivatives of

bisphenol- A-diglycidylether.

Table 8. Fonnaldehyde* detected in AH 26" and ~ 2 @ samples at different setting times f adapted fiom Sphgberg et al., 1 993)

I Setting Time I AH 26@ I ~2~

Freshl y mixed

12 hr

24 hr

2 days

7 days

Kaplan and associates (1999) concluded that AH 26@ possessed antimicrobial

properties and was capable of inhibiting the growth of Candi& afbicans, Streprococcz~s

rnzltans. Sfaphyfococus aurem and Actinomyces israeliz after a contact period of 20 to 40

days. The antimicrobial effect of AH 26@ was attributed to the release of formaldehyde

during this time period.

Briseiio and Willershausen (1991) compared the cytotoxicity of AH 26@, ~iaket@ (a

polyketone sealer; ESPE, Seefeld, Germany). and Lee ~ndo-~i l l " (a si1 icone rubber sealer;

Lee Pharmaceuticals, El Monte, CA) sealers by rneasuring the incorporation rates of 14c-

labelled leucine in human gingival fibroblasts to synthesize protein after direct contact with

these test materials. When set for 24 and 48 hours prior to testing, AH 26" produced the

O. 12

0.27

Powder cornponent

Resin component

*

83 5 .O0

1740.00

1.50

22.26

13.39

1020.00

73 10.00

149.00

*ions x 106/mg of material

None

None

Overload

None

most severe cytotoxic reactions. Based on the findings that no fibroblast recovery could be

measured for AH 26@ set for two days, the investigators concluded that the release of toxic

products occurred for a longer time period than the reported setting time for this material.

Although in vitro toxicity data are relative and not directly transferable to iri vivo

situations (Arenholt-Bindslev and Horsted-Bindslev, 1989) the in vitro cytotoxic findings

of AH 26" are supported by case reports of nerve damage foliowing endodontic treatrnent

with this sealer which, fortunately, could be corrected by either surgical decompression of

the affected nerve (Spielman et al., 1981) or extraction of the causal root-filled tooth

(Barkhordar and Nguyen, 1985). Despite the low levels of FA released by this material,

Koch and associates (2001) recommended the use of FA-free endodontic sealers in

individuals with FA allergy.

Glass Ionomer Cernent

Dunng the 1960s Wilson and Kent conducted the initial investigations considering

glass ionomer cement (GIC) and its dental applications (1972, 1973). The first to suggest

the use of GIC as a potential root filling material was Pitt Ford (1979), who performed an

itt vitro dye penetration study comparing single-rooted, earacted teeth obturated with

experimental GIC sealers and a single gutta-percha or silver cone. He concluded that GIC

material (Le. ASPA IV) provided a satisfactory seal and recommended changes in

formulation to improve clinical handling characteristics (e.g. longer working time). Many

chemical modifications have since been made to improve upon the chemical, physical,

clinical and biological properties of this restorative matenal (McLean, 1988; Smith, 1998).

GIass ionomer cernent chemistry

Aluminosilicate glasses with added calcium and fluoride ions (based on the SiO2-

Al203-Ca0 or Si02-Al203-CaF2 system) are used in al1 commercialiy available GICs. The

glass particles are made by fùsing the ingredients at temperatures ranging between 1200 to

1500 OC and then shock-cwiing the glasses into a coarse frit with water. The glass

particles are then milled to the desired size, typically diameters of 45 prn for restorative

materials and 1 5 Fm for luting cements (Nicholson, 1998).

Glass ionomer (or polyalkenoate) cements in their set form are a polysalt matnx

embedded with residual glass particles as the result of an acid-base reaction between

powdered fluoroaluminosilicate particles (the glas component) and an aqueous

polyacrylic acid. This interaction involves the release of metal cations from the glas

component which in tum facilitates cross-linking between chains of the polyacid. The

extent of cross-linking is a detenninant factor in the cement properties, as well as the glass

volume fraction and the molar mass, reaction time and concentration of the polyacid (De

Barra and Hill, 1998). The silicon and phosphorus elements of the glass component have

also been shown to form a hydrated inorganic silicate/phosphate network that

interpenetrates with the cross-linked metal polyacrylate matrix and is thought to increase

the insolubiiity and compressive strength of the material over tirne (Wasson and

Nicholson, 1991).

Moditications in GICs used today include the incorporation of different polymers

( e g . acrylic and maleic acid), metal-reinforced and cermet-modified glass components, as

well as the addition of monomers and initiators with photochernical polymerization

capabilities (Nicholson, 1998). Glass ionomer cements with a "water-hardening"

formulation have a powder component of fluorosilicate glass, tartaric acid and polyacrylic

acid (e.g. ~etac-cemg. ESPE Dental-Medizin GmbH & Co. KG, Seefeld, Germany; Table

9). Hydroxyethylmethacrylate (HEMA) can be included in the liquid water component to

increase the setting time relative to conventional GICs (Saunders et al., 1992).

Table 9. The composition of ~etac-cem@ (adapted fiom ESPE Dental-Medizin product dossier)

The addition of 540% (+)-tartaric acid (e-g. ~ e t a c - ~ e m " and ~e tac@-~ndo , Table

10) improves the handling properties (extends the working time and sharpens the later

setting stage) (Crisp et a/., 1975) and compressive strength of the material but the actual

mechanisms by which these occur is unknown (Nicholson, 1998).

Recently there has been a shifi in the use of commercially available GICs, fiorn the

traditional acid-base reaction setting system to one utilizing a light-cured polymerization

reaction similar to that in resin composite materials. These polymer-reinforced GICs

utilize dentine-bonding adhesive systems and are formulated with the intention of

broadening the clinical applications of GICs while retaining the anticariogenic and

biocompatibility properties (Smith, 1998).

Powder Component sodium-calcium-aluminium-lanthanum- fluorosilicate glass

vacuum dried copolymer and acrylic and maleic acids

pigments

Liquid Component

tartaric acid

water

Table 10. The composition of ~ e t a c @ - ~ n d o Aplicap (adapted fiom ESPE Dental Medizin product dossier)

Powder Component

1 calcium wolfiamate I tartaric acid

Liquid Component

calcium-aluminum-lanthan-fluorosilicate glass

silicic acid water

acrylic acidmaleic acid copolymer

Biocompatibility of glass ionomer cements

The health of oral tissues may be affected by the formation and release of water-

soluble components into saliva, as well as by the direct interaction of the GIC material with

adjacent tissues (Smith and Williams, 1982). Due to their broad range of clinical

applications, the GICs should be classified as materials in permanent contact with living

tissues, such as the periodontal ligament, gingiva and alveolar bone (Leyhausen et al.,

1 998).

The biocompatibility of glass ionomer cernent has been well documented in previous

literature, including experiments involving GICs specifically formuiated as root canal

sealers (Jonck et al., 1989a & 1989b). 1n vitro cytotoxicity of ~ e t a c @ - ~ n d o to a ce11 line of

baby hamster kidney tibroblasts (BHK2 1/C 13) was determined to be very low (Beltes et

al., 1997). Animal studies investigating the biological safety of ~ e t a c @ - ~ n d o have

reported a mild inflamrnatory response at 5 days after subcutaneous implantation in rats

and total healing afier 120 days (Kolokuris et al., 1996). These findings support those of

pigments benzoic acid

Blackman and associates (1988), who implanted a silver-containing GIC into the soA

tissue and bone of rats. An initial inflammatory response was produced which subsided

afier one month. Bone surrounding the GIC material was found to have regenerated

rapidly, even forming directly upon the GIC surface.

To take advantage of their proven osteogenic characteristics, glass ionomer

cements are being considered for use in orthopedic surgery. Oliva and associates (1996)

tested the response of human osteoblastic ceils to five commercial dental GICs in vitro.

Four of the GICs tested were deemed biocompatible, showing vital cells adhering to the

cement materials. Using proton magnetic resonance analysis, cytotoxicity of the fifih GIC

was attributed to leaching of at least two components its polyacid phase: 1) 2-

hydroxyethymethacrylate (HEMA) and 2) an unidentified acidic species. Hypersensitivity

to dimethacrylates (e.g. HEMA) (Kanerva et al., 1993) and oestmgenic effects of bis-

phenol A products in these GIC/resin modified restorative materials (Olea et al., 1996) are

biocompatibility issues being currently investigated.

Glass ionomer cement bonding to dentin

Pnor to the development of GICs, there were no materials specifically

manufactured as endodontic sealers capable of chemically bonding to tooth structure

(Saunders et al., 1992). The adhesive bonding of GIC to natural tooth structure has been

shown to increase the fracture resistance of teeth when used to restore cavity preparations

as a base material (Trope and Tronstad, 1991) and also when compared to silver arnalgam

as a restorative material (lagadish & Yogesh, 1990).

The mechanism for GIC bonding to enamel is the result of ionic and polar forces

(Lin et al., 1992). Dunng setting, the carboxylate anions of the GIC compete for covalent

cross-linking sites on both the inorganic dentin matrix surface (Wilson et al, 1983), which

consists mainly of hydroxyapatite (C~IO(PO&(OH)~) (Trowbridge, 2001), and also the

organic collagen component (Friedman et al, 2000).

The setting stages of the GIC are the result of an acid-base reaction:

1) Stage 1 : The release and migration of glass ions after acid aîtack.

2) Stage 2: Binding of cations (e.g. Ca', Ai') to polyanions (of the polyacid) resulting in the precipitation of salts and subsequent gelation and hardening. If, during the gelation stage the GIC is exposed to water, cations will be washed out and resuit in the formation of a weakened cernent.

3) Stage 3: Hydration of salts resulting in increased strength.

Additional stability of the GIC bond is provided by mechanical interlocking of the

cement extending up to 48 pn into the dentinal tubules (Weiger et al., 1995). Saunders

and associates (1992) used SEM analyses to show a close adaptation of GIC into the

dentinal tubules of roots treated with 40% citric acid to remove the smear layer prior to

obturation with gutta-percha and GIC sealer. They speculated it was the hydrophiiic

HEMA liquid component that facilitated favorable flow characteristics into the

demineralized tubules (diameter 2-4 pm) since the glass particles (ranging 8-45 pm) would

be too large to enter. These investigators also hypothesized that chernical bonding occurs

between the GIC and gutta-percha cones by chelation of zinc ions by the polyacrylic acid,

resulting in the formation of salt bridges.

Coronal dentin has a higher density of dentinal tubules than that found in the root

(Pashley et al., 198 1 ) but the resulting increase in intertubular dentin of the root and its

effect on the bonding characteristics of GIC is unknown. One of the few studies

investigating the adherence of GIC specifically to root dentin was conducted by Berry and

Powers (1994), who hypothesized that the lower dentinal tubule density would yield an

increased GIC bond strength. Factors involved in GIC bonding t o dentin were determined

to include: the cohesive strength of the cernent, type of dentin surface, dentin surface

treatment, and the ionomer-dentin interface. The shear bond strengths were greater to

radicular dentin than coronal dentin. The conditioning of dentin with either 25% or 40%

polyacrylic acid seemed to increase GIC bond strengths compared t o that with dentin

undergoing no conditioning treatment. This finding is in agreement with Stanley (1990),

who described acid etching the canal dentin pnor to placement of GIC sealer as a process

which removes surface contaminants, allowing increased ionic exchange and facilitat ing an

improved bonding between the cement and tooth structure.

The chemical bonding of GIC to dentin o f the r w t canal system should be reflected

by a decreased leakage in roots filled with this material. 112 vitro studies by Miletic and

associates (1999) compared leakage in teeth obturated with gutta-percha cones and

di Rerent endodontic sealers (i ncluding ~ e t a c @ - ~ n d o and AH 26@ (silver-free)) using a

fluid transport mode1 previously described by Wu and associates (1993). Aithough not

statistically significant, the least leakage was observed in teeth obturated with the ~ e t a c @ -

Endo cernent.

During instrumentation of the root canal wall a smear layer is produced which is

comprised of dentin as well as tissue debris (both necrotic and vital, including remnants of

odontoblastic processes, pulp tissue and bacteria) (McComb and Smith, 1975). The effect

of this smear layer on GIC binding to dentin is not fully understood. Lalh and associates

(1999) found the presence of a smear layer to significantly enhance GIC bonding strength

to bovine dentin. ûthers believe it may decrease dentin permeability and limit diffusion of

bacterial metabolites fiom the root canal to the external root surface, potentially reducing

the occurrence of subsequent penradicular pathosis (Galvan et al., 1994). Bacterial

penetration and coionization of the dentinal tubules resulting in reinfection of the root

canal space may also be reduced by leaving the smear layer intact (Drake et al., 1994).

Friedman and associates (1992) compared the eficacy and time required to retreat

root canals using either hand or ultrasonic instrumentation in teeth obturated with gutta-

percha and one of three sealer types: ~oth ' i ' 80 1, ~e tac@-~ndo, or AH 26'& (with silver).

Ultrasonic instrumentation was significantly faster than using hand instrumentation for al1

sealer types. The amount of debris lefi on the canai walls was observed to be greatest in

teeth obturated with ~e tac@-~ndo, possibly due to its dentin-binding properties (Friedman

el al., 1 993). They concluded that retreatment of ~ e t a c @ - ~ n d o was facilitated by use of the

ultrasonic technique and the time required was comparable to that required for teeth

containing AH 26@ sealer.

Resistance to root fracture

The amount of remaining sound tooth structure is probably the single most

important factor related to the strength of an endodontically treated tooth (Trabert et al.,

1978). Although coronal reinforcement is possible through restorative bonding techniques,

there has been relatively little research in the application of adhesive materials to reinforce

the root canal systern. Glass ionomer cernent sealers are being developed with the potential

to increase resistance to root fiacture.

The prognosis of a tooth with vertical root fiacture is extremely poor (Selden,

1996). Root fracture is a potential outcome following endodontic treatment (Sorenson and

Maninoff, 1984)' in part due to structural weakening during caries removal, endodontic

procedures (Le. access preparation' instrumentation of the canal) (Silver-Thom and Joyce,

1999) and preparation for a definitive restoration (Le. post and core prior to cast crown

fabrication) (Standlee et al., 1 972). Silver-Thorn and Joyce ( 1 999) concluded that the

stresses created dunng penetration of the spreader instrument used to condense gutta-

percha may be a primary cause of vertical root fracture. According to Pitts and Natkin

(1983), a vertical root fracture extends throughout the entire dentin thickness fiom the roat

canal to the periodontium and can involve any part of the mot. Locdized bone loss and

periodontal pockets can subsequently develop, forcing the patient to consider either

surgical resection of the fi-actured root or extraction of the tooth altogether.

The mandibular incisor may be the tooth at greatest nsk for fracture during lateral

condensation of gutta-percha cones, due to its narrow root morphology and thin root dentin

(Lertchirakarn et ai., 1999). An il1 vdro laboratory study was conducted on extracted

mandibular anterior teeth to determine if ~e tac@-~ndo, used as a mot filling material,

affected resistance to root fiacture (Johnson et al., 2000). No significant improvements in

fracture resistance occurred, regardless of whether the root was lef? unfilled or obturated

with GIC, composite resin, or gutta-percha and a ZOE-based sealer (Table 1 1).

The investigators suggested that significant increases in root strength could only be

attained if sufficient amounts of restorative material can be used. Thin-walled, immature

roots represent such a clinical situation where increased amounts of restorative material

can be used to potentially reinforce the remaining root stnicture.

24

Table 11. Force to fiacture in roots obturated with different materials (adapted corn Johnson et al. 2000)

1 Instmmented but not obturated I 43.77

Croup (n = 15 for each)

Mean Force at Fracture (kg)

Lateral condensation (gutta-percha + Roth's sealer)

44.50

~e tac%ndo (with single gutta percha cone)

41.27

et aca-~ndo (canal pre-treated with 25% polyacrylic acid)

44.47

ICetaclv-~ndo (canal pre-t reated with 17% EDTA)

Standard Error

46.07

Bonded composite resin

Pene and others (2001) corroborated the findings of the previous study. Teeth

mimicking immature, nonvital maxillary incisors were produced using an engineering twist

drill (3 mm diameter) to enlarge the canal space of extracted maxillary central incisors. An

Instron Testing Machine was used to evaluate the fracture strength of teeth with their canal

space: 1) left unobturated, 2) filled with dentin-bonded composite resin (Prisma VLC'

Hybrid, Dentsply Co., Konstanz, Gennany), o r 3) filled with dentin-bonded composite

resin and. reinforced with ribbon made of a fiber composite laminate (connecta, Kerr Co..

Romulus, MI). Significant differences resulted in fracture load values for the three test

groups (one-way ANOVA, pK0.003). The control (unobturated) group ftactured at the

lowest loads (mean = 3 1 .O8 kg; standard deviation = 3.39 kg) while the composite-filled

37.07

mots had the highest resistance to fracture (mean = 51.00 kg; standard deviation = 3.65

kg). The nbbon-reinforced composite filled group fiactured at a lower mean force (37.93

kg; standard deviation = 4.82 kg) than those teeth filled with only composite.

Investigators attributed this unexpected finding to a lack of chemical bonding

between the ribbon and the resin. The volume occupied by the reinforcing ribbon instead

acted as a void in the composite. The resultant reduction in composite used to restore the

root created a concomitant reduction in root fracture strength. Taken together, the findings

of these two studies demonstrate that the amount of remaining mot structure relative to the

volume of root filling material has an effect on whether or not significant root

reinforcement occurs a€ter endodontic treatment.

Clinical case reports exist describing the use of GIC to reinforce root-fractured

teeth (Stewart, 1990; Trope and Rosenberg, 1992). An il? vitro study resulted in a higher

fracture resistance in teeth obturated with gutta-percha and GIC sealer (~etac@-~ndo)

when compared to teeth obturated with conventional gutta-percha and ~ 0 t h ' ~ ~ cernent or

instrumented but lefi unfilled (Trope and Ray, 1992) (Table 12).

Table 12. Resistance to fiacture of endodontically treated mots (adapted fiom Trope and Ray, 1992)

Obturated with gutta-percha and Roth's

sealer

mean load ( w

standard deviation

t

1

Uninstrumented cmals

Obturated with gutta percha +

GIC sealer

176.4

52.19

hstrumented canals (but not

abturated

158.5

41 .29

105.5

40.5 1

1 15.6

42.99

Trope and Ray (1992) advised the use of only a single gutta-percha point to

facilitate endodontic retreatment procedures. By eliminating the need for laterally

condensing additional gutta-percha points d u h g obturation, vertical root fractures could

be avoided.

Antibacterial properties of glass ionomer cernent

The antibacterial potential of GIC is related to its fluoride-releasing capacity, low

pH levels during the setting process, and the presence of certain cations in some restorative

preparations (e.g. strontium and zinc) (Herrera et al., 200 1). The release of fluoride and its

cariostatic properties, however, is the focus of most research conducted on this material.

Metabolic inhibition of cariogenic bacteria and remineralization of enamel and dentin

tissues are examples of mechanisms by which fluoride exerts its anticariogenic effects

(Forsten, 1998). The release of fluoride is a dynamic phenornenon related to the GIC

structure, the external environment, and the time fkorn mixing. Freshly rnixed GIC has

been shown to release three to ten times more fluonde than that released by the material

after it has set for three days (Forsten, 1991). M e r approximately two years, fluoride

release decreases to a constant level (about 1 ppm), as recorded from GIC test specimens

lefi in ninning water for 29 months (Forsten, 1991). Unintentional voids lefl between the

tooth structure and filling material will have a high concentration of fluoride (especially

fiom the initial "burst" release of fluoride) which will inhibit the viability of cariogenic

bacteria and also harden tooth structure by induction of remineralization (or even

hypermineralization) of dentine and enamel (Forsten, 1998).

Since the constant release of fluoride from GICs subsequent to the initial "burst" is

assumed to be quite low, it is desirable that these materials show an ability to acquire

fluoride fTom extemal sources, and in eflect "recharge" themselves to create a sustained

and dynamic fluoride-releasing system. Forsten (1991) demonstrated the ability of GIC to

reiease increased tluoride concentrations afler being treated, or "recharged, with a 50 ppm

fluoride solution. This effect was not observed with composite resin specimens used for

comparison. Laboratory studies (Forsten, 1998) suggest a constant level of fluoride release

as long as the GIC filling material remains intact and within the tooth cavity preparation

(exceeding 8 years). Of the ten GIC materials tested, ho tac- il" (ESPE/Premier,

Nomstown, PA) showed the highest release of fluoride while polyacid-modified

composites (e.g . ~ y a c t " , Dentsply, Konstanz, Germany) exhi bited no "burst" effect at d l .

The anticariogenic property of GIC has as much relevance to the radicular tooth

structure as it does to coronal areas. Infection may develop within the root canal system

when coronal ingress of bactena andor their nutrients occurs, so the benefit of a root

filling matetial providing resistance to microorganisms is obvious. An increased fluoride

concentration was detected in the dentin of roots filled with gutta-percha and GtC

(vitrebond@, 3M Dental, St. Paul, Minnesota) &er an in vitro period of three months,

using scanning electron microprobe analyses (Saunders et al., 1992). A direct contact

inhibition test of four different GICs was conducted by Herrera and associates (2001) who

found that each of the cements demonstrated antibacterial activity but to varying degrees.

vitrebond" was the only material, for instance, to inhibit al1 Laclobacillus strains tested.

Most studies have concluded that ~etac"-~ndo only exerts significant antibacterial

activity when fieshl y mixed. Abdulkader and associates (1 996) found fieshl y mixed

~e tac@-~ndo sealer to in hi bit the growt h of Porphyomo)tas gingr valis, Capnocytophaga

ochracea, and Peptos~rep~ococcus micros in an in vitro direct contact test. Shalhav and

associates (1997) utilized two in vitro assays (agar difision and direct contact tests) to

evaluate the antibacterial properties of ICetacS-~ndo when compared to ~ 0 t h ' ~ ~ cernent.

The agar diffusion test resulted in freshly mixed ~ e t a c @ - ~ n d o samples exhibiting

significantly greater antibacterial activity than ~ 0 t h ' ~ ~ cement ( ~ ~ 0 . 0 5 ) . In the direct

contact test, only the fieshly mixed ~e tac@-~ndo exhibited antibacterial activity, while

samples set for either 24 hours or 7 days showed no such activity. It was concluded that

this GIC possesses a potent, diffisable, antibacterial activity that is significantly

diminished at 24 hours from mixing.

Many studies investigating the antimicrobial activity of GICs do not suggest them to

be effective against known endodontic pathogens. Kaplan and associates ( 1999) showed

~ e t a c ' ~ - ~ n d o to have little effect on inhibiting the growth of Candida albicam,

Streptococcus mutans and StaphyIococçus aurelrs afier contact periods of 2, 20 and 40

days. An in vitro investigation on the antibacterial effects of several endodontic sealers,

including et ac@-~ndo, on bovine dentin infected with Enterococms faecalis was

performed by Heling and Chandler (1996). Their findings indicated that at 24 hours from

mixing, ~ e t a c @ - ~ n d o exerted antirnicrobial effects no greater than saline controls.

Clinical studies using glass ionomer cernent as a root filling material

In one of the few clinical investigations using GIC as a root f i lhg material,

Friedman and associates (1995) used ~ e t a c @ - ~ n d o as a sealer in roots obturated with a

single cone or laterally condensed gutta-percha. Of the 378 teeth that were assessed

between 6 to 18 months postoperatively (78% recall rate), complete penradicular healing

occurred in 78.3% and incomplete healing in another 15.6% of the study sample. The

absence of a coronal restoration at the time of recall examination was related to failure to

heal (significant at p<0.03). This finding suggests that in the absence of a coronal

restoration, ~ e t a c @ - ~ n d o is unable to prevent microbial ingress into the canals, resulting in

failure of the endodontic treatment.

Zeolites in dentistry

General structure and characteristics

Zeolites (Gr. zein, "to boil"; [zthos, "a stone") are so named to describe the visible

loss of water observed when natural zeolites are heated. This property (intumescence)

demonstrates the high degree of mobility of water through zeolite structures (Dyer, 1988).

The ease with which water molecules are not only lost, but also regained, from zeolites

accounts for their well-known dessicant properties.

Although zeolites are natural minerals found in many parts of the world, most used

for commercial purposes are synthetically produced. They are microporous, crystalline

solids with well defined structures containing aluminum, silicon and oxygen in their

regular fiamework. The aluminum and silicon atoms share oxygen atoms to form a

coordinated tetrahedral arrangement ( e g Si04 and Aloi). Void spaces (cavities or pores)

ranging from 3 to 10 A are found within the zeolite frameworks that can host cations,

water, or other organic molecules (see Figure 2). The aluminosilicate fiamework

arrangement results in a net negative charge that is balanced by the cations present dunng

zeolite synthesis. These cations are highly mobile and can be exchanged for other species.

The size of the apertures produced within the zeolite fiamework account for their ability to

selectively take up certain molecules and exclude others based upon their larger

dimensions, a property called "molecular sieving" which is unique to zeolites.

Figure 2. Iliustration of a hexane rnolecule containeci within a zeolite structure (source: http://tnm.cycl. phys.tue.~noordhoeWzeolite.html)

Aside fiom the structural characteristics of a zeoiite, cation exchange is also

iduenced by (Breck, 1984):

1) The type of cation species cation size, both anhydrous and hydrated, and cation charge.

2) Temperature. 3) The cation species concentration in solution. 4) Theanionspeciesassociatedwiththecationinsolution. 5 ) The solvent (usuaUy an aqueous solution).

Approximately one miilion tons of zw lite is produced worldwide each year, mainly

for application in the areas of ion exchange, catalysis and separation technology. An

example of their commercial application is their use as water-soflening agents in an ion-

exchange rnethod termed "zeoiite process". Zeolite material has ken successfülly used to

eliminate radioactive wastes (Elizondo et al., 2000) and also as a suitable carrier for

anthelmintic drugs @yer et al., 2000).

~eomic@: a silver-containing zeolite

The Sinanen Zeomic Company, Ltd. (Nagoya, Japan) developed Zeomica in 1983

as synthetic ("A" type) zeolite in which silver ions have been exhanged for sodium ions.

The United States Food and Dmg Administration (U.S.F.D.A., under the Federal Food,

Drug, and Cosmetic Act) approved it in June 2000 as an antimicrobial additive to be used

in al1 types of food-contact polymers (also known as a "food contact substance"). The

manufacturer has since aimed the development of ~eomic@ as a food, cosmetic and

pharmaceutical additive with long-lasting antimicrobial efficacy through the release of low

levels of silver ions (U.S.F.D.A. and Sinanen Zeomic Company, Ltd. websites).

The antimicrobial activity of silver-containing zeolites has been established and

applied in fields as diverse as animal foods to synthetic detergents. Used to mat medical

catheter tips, silver was shown by Schoemer and associates (1999) to effectively inhibit the

growth of various bacterial species iii vitro. The mechanism by which silver ions (Ag')

exert their antimicrobial effects is not fùlly understood (Feng el al., 2000) but is probably

related, in part. to their strong binding affinity to electron donor groups containing sulfur,

oxygen and nitroser.. These elements are commonly found on bactenal ce11 surfaces and

biological molecules such as thiols aminoimidazoles, carboxylates, and phosphate groups

(Lehninger et al., 1993). Another property of silver cations likely contributing to its

antimicrobial properties is its ability to displace other essential metal ions, for example,

copper (cu23 and zinc (Znt) (Smith and Williams, 1982).

Some of the sites of action by which silver ions exert their antimicrobial effects

have been elucidated and show to disrupt processes at both the ce11 membrane and

cytoplasmic levels:

a) Silver ions affect processes of the respiratory chah in Escherichia coli, possibl y between cytochromes b and a2, and/or NADH-succinate and flavoprotein.

b) Daughter cell separation of this bacterium is inhibited by silver ions.

c) Ag' binds to microbial DNA molecules, causing them to condense and lose their ability to replicate.

d) Ag' can inhibit many enzymes including ATPase, urease, &galactosidase and various dehydrogenases.

The most important interaction through which silver exerts its antimicrobial action

is uncertain, but this effect probably occurs as a result of interference with a number of

vital ce11 surface and cytoplasmic processes (Feng et al., 2000; Smith and Williams, 1982).

Because of the multitude of interactions between silver cations and vital cellular processes,

it is dificult for bacteria to develop resistance against silver-containing compounds

compared to other antimicrobials with specific sites of action (e.g. penicillin inhibition of

peptidylglycan ce11 wall formation). Resistance to silver has been documented for various

bacteria (including Escherichia coli, Salmo~~ella ryphmt~rimn. Pseudomonad) (Russell,

1994) through the ability to exclude andor efflux silver, or possibly by an intracellular

detoxification mechanism (e.g. metallothionein) (Slawson et al, 1992). The emergence of

resist ant bacterial strains i s most likel y to occur in environment s with high concentrations

of silver (e.g. silver mines, hospital bum units, photographie processing plants) where

selective pressures would favor t heir establishment (Williams, 1 990). Reports of silver

resistance in rnicroorganisrns associated wit h pathosis of endodont ic origin, however (to

the authors knowledge), do not exist in the present literature.

~eomic@ AJ-1OD (Shinagawa Fuel Co. Ltd., Tokyo, lapan) is a commerckdly

available zeolite containing a core of silver ions (Niira et al., 1 996; see Figure 3; Table 1 3).

Figure 3. Scanning electron micrograph of ~eomic@ particles (shown lefi) which cm be milled to produce an odourless, white powder for commercial applications (fiom the Sinanen Zeomic Co., Ltd. website)

~eomic" is able to continuously release silver ions into water at concentrations

capable of providing long- term ant imicro bial act ivity (approximately 1 0 ppb) and t hat are

not hannfiil to tissue c e b (Breck, 1974). Several investigations have been conducted to

ve* the biological safety of 2eomico. An Ames Test (Japan Food Research

Laboratories, test report no. N A 39 120 1 55-2) suggests no mutage& activity. Also, there

was no suggestion of primary skin irritation when 2eom. i~~ was tested using the Dmize

method (Nomura Bio-scientific Researc h Institute, test report no. NRILS87-2209). The

dose at which ~eornic@ kills 50% of test anirnals (rats) (LDsd5000 mg ~eomic@/k~ animal

weight) suggests this material to have a very slight acute toxicity. Sodium chioride, by

Table 13. Physical and chemical charactenstics of ~eomic" (adapted from Matsuura et al., 1997 and the Shinanen Zeomic Co., Ltd. website )

Structural fornula

1 Weight percentage of components

Shape

Poms size

1 Specific gravity

Comparative surface area

( Average panicle size

1 Specific heat

1 Heat resistance

1 Acidity resistance

Alkalinity resistance

MX~/,O*M~O~*YS~OÎ-ZH~O M: cation (Ag', 2n2', etc.) X, Y and Z: mole fiaction of each component AgT:2.5 wt?!; ~ n " : 14.5 wt%; m 4 : 2.5 wt%; H20: 16- 18 wt% Odourless, white, fine powder

0.6-2.5 (pm)

0.26 (caYg)

550 (OC)

cornparison, has an LDSo of 3000 mgkg body weight (Shinanen

booklet). The results of these studies indicate 2eornica to be

material.

Zeolite incorporation into dental materials

The clinical application of zeolites in dental matenals is

~eomic@ Co. information

a biologically acceptable

a relatively new concept.

2eornicm has been combined with a commercially available tissue conditioner used to line

dentures at concentrations ranging fiom 2 to 5% ( d w t ) and was shown to reduce growth

of C d & albicans in a dose-dependent manner (Nikawa et al.. 1997). Matsuura and

associates (1997) incorporated 2eomicm into the powder (concentration 2% wt/wt) of five

commercially available denture tissue conditioners to determine its antimicrobial effects on

C. albicans and nosocomial respiratory infection-causing bacteria S~aphyiococ~~s uzwezds

and Pseudomonas aemginosa. They demonstrated that antimicrobial effects against t hese

three pathogens occurred in vitro after four weeks immersion in saliva. The findings of

these two studies suggest that the combination of an antirnicrobial-containing zeolite and

denture tissue conditioner would be a potential aid in denture plaque control.

A potential temporary dental filling material consisting of a zeolite agent carrying

both silver and zinc ions (~actekille?, Kanebo, Japan) was incorporated with Si02 filler

and urethane acrylate monomer paste in varying amounts (from 5/55 to 30/30 wtY0). A

dye penetration test was used to measure growth inhibition of four oral bacteria

(Streptococclrs mzifans, S. milis, S. salivnriz~s, and S. sangrtis). There was prominent in

vitro antimicrobial activity against rnuzans and S. mitis and also a measurable release of

silver and zinc afier four weeks. Although the cation release was determined to be dose-

dependent, a higher ratio of zeolite content did not result in an increased antibacterial

activity (Hotta et al., 1998).

Morishita and associates (1998) evaluated the inhibitory effects on plaque

formation when human subjects used a silver-containing zeolite mouthrinse (concentration

3% W/W) for five days. There was a significant reduction in plaque formation in subjects

using this zeolite agent compared to wntrols, suggesting another application in which

zeolites could inhibit bacterial colonization.

Kawahara and associates (2000) evaluated the antimicrobial effects of silver zeolite

against oral bacteria under anaerobic conditions. The minimum inhibitory concentrations

(MICs) of silver zeolite ranged between 256-2048 pg/mL (equivalent to 4.8 - 38.4 pg/mL

silver ions). These results suggest that silver-containing zeolite can impart antimicrobial

activity to dental materials when exposed to anaerobic conditions encountered in the oral

environment (e.g. a periodontal pocket or the root canal system).

Composite resin dental matenals, which contain polymerizable components

reinforced with filler particles (organic or inorganic) have not been incorporated with

antimicrobial zeolites for development as a root canal sealer. One reason is that shrinkage

of the composite resin occurs as it polymerizes, creating void spaces between the

restorative material and the tooth structure into which microorganisms can ingress and

reinfect the root canal system. When exposed to the oral environment, the relative

insolubility of the resin matenal would not facilitate ion exchange activity within the

zeolite structures, substantially reducing the antimicrobial potential of the zeolite agent.

ZUT: a zeolite-containing glass ionomer cernent endodontic sealer

A zeolite-containing, GIC-based root canal sealer (ZUT) is being developed at the

Faculty of Dentistry, University of Toronto. By definition, ZUT is a GIC incorporated

with silver-containing zeolite material to be used as an endodontic root-filling material.

The porous zeolite ceramic framework can be incorporated into the glass powder

component of GIC and is capable of forming inorganic bonds with the polyacid component

during the setting reaction (unlike composite resin material). The silver ions contained in

the pores of the zeolite fiamework cm be leached out of the cerarnic structure and provide

antimicrobial activity (Patel et al., 2000).

To determine the effect of ~eomic@ on the dentin-binding characteristics of GIC,

Lalh and associates (1999) compared the shear bond strength to bovine dentin of ZUT

0.2% (containing 0.2% Zeomic* Wwt glass content) to that of ~ e t a c @ - ~ n d o and the GIC

component of ZUT (in this case, KT-308; GC Corporation, Tokyo, Japan). The influence

of dentin exposure to various irrigants (distilled water, 2.6% NaOCl, or 17% EDTA

followed by 2.6% NaOCI) prior to contact with the test materials was also evaluated in

vitro. Results suggest ZUT and KT-308 had significantly higher shear bond strength values

than ~ e t a c - ~ n d o (2-way ANOVA, p<0.0001) and that the presence of a smear layer

enhanced GIC bonding (pc0.02). The addition of 0.2% Zeomicm did not affect the shear

bond strength of the GIC component of ZUT alone.

Patel and associates (2000) compared the antimicrobial effects of ZUT sealer

incorporated with three different concentrations of 2eomica> (0.2, 2.0, and 20 % wt/wt

ceramic component) on E. faecalis in vitro. Even after 12 weeks immersion in Brain Heart

Infusion broth (BHI; Difco, Detroit, Ml), al1 concentrations of the zeolite-containing GIC

samples effectively suppressed bacterial growth in a direct contact inhibition test. These

results suggest that even at 0.2% 2eomicB concentration. ZUT (ZUT 0.2%) provides a

sustained, long-term antimicrobial activit y.

McDougall and associates (1999) incorporated KT-308 GIC with 2eornica (2%

W/W concentration = ZUT 2%) to test its ability to inhibit the penetration of Enterococns

faecalis into root canal systems in v&o. There were no significant differences in the

incidence of bacterial penetration in canals obturated with ZUT 2% (and a single gutta

percha cone) compared to those filied with KT-308 GIC alone (X2 and Fisher's exact tests;

two-tail; p>0.05). These results suggest that the addition of 2eomica (at 2% 2eomicm

w t h t GIC glass) did not adversely affect the sealing ability of GIC. An unexpected

finding was the significantly higher incidence of bacterial penetration in roots filled with

ZUT 2% compared to those obturated with ~ e r r @ PCS (a zinc-oxide endodontic sealer

containing silver). Presumably, the unique dentin-binding characteristics of GIC sealers

(Le. ZUT) should result in superior resistance to bactenal penetration compared to

convent ional ZOE-based endodontic sealers (Le. ~ e r r @ PCS).

Padachey and associates (2000) conducted a sirnilar bacterial penetration study

comparing root canals filled with either KT-308, ZUT containing 0.2% zeolite (wt/wt GIC

glass), or AH 26@ sealer. The incidence of E. faecalis penetration did not differ among the

three test groups (2 and Fisher's exact tests; two-tail; p > 0.05). The use of a single gutta

percha cone resulted in decreased bacterial penetration, regardless of the type of sealer

used to obturate the canals. These results support those of McDougall and associates

(1999), suggesting the addition of 0.2-2% 2eornicm does not adversely affect the sealing

ability of GIC. The results of these two in vitro studies however, do not suggest ZUT

offers superior eficacy in preventing bacterial ingress into the root canal system.

Results fiom the previous two studies (MacDougall et al., 1999; Padachey et al.,

2000) put the ability of ZUT to seal the root canal system in question but results of in vitro

leakage studies must be interpreted with caution. A lack of agreement oflen exists among

studies using different methods evaluating the sealing effectiveness of root filling materials

(Barthel et al., 1999; Pommel et al., 2001). Dye leakage is the technique most often used

to evaluate the sealing ability of a material due to its simplicity. Other means of evaluating

leakage into the root canal system include bacterial ingress, electrochemical methods, fluid

filtration, and radioisotope labelling (Pommel, 200 1).

Barthel and associates (1999) found that no correlation existed between a bacterial

leakage and dye leakage study in which 37 obturated teeth leaked to bacteria, 18 leaked to

dye, and 12 teeth leaked to both bacteria and dye. Pomme1 and associates (2001)

conducted an apical leakage study of three obturation techniques using three evaluation

methods (each used successively on the same teeth; 1-way ANOVA with Duncan's

pairwise t-tests). Results are summarized in Table 14; there was an obvious lack of

comelation in leakage rankings for the obturation materials when different testing methods

were employed.

Table 14. Apical leakage in vitro of three obturation techniques using three evaluation methods (n = 12 for each group) (adapted fiom Pomme1 et al, 2001)

Leakage Ranking

Best

Intermediate

Worst 7

Fiuid Filtration 1 Electrochemical Method

*vertical 1 no statistical condensation -1 ,h,'tEo"i::inoBn

Dent spl y, Milford,

technique

1 techniques

Dye Penetration

No statistical differences

between vertical condensation and

single cone techniques

*denotes significance at p = 0.04

The lack of correlation might be attributed to the criteria used to establish leakage

differences between the techniques, rendering findings clinicall y irrelevant . The

investigators found these results "disturbing and raises doubt on the previously published

papers" investigating i r ~ vitro root canal leakage.

For fbture leakage studies, Pomme1 and associates (2001) recommended that

several methods of evaluation are employed and several data sets are recorded prior to

drawing any conclusion. ZUT, for instance, may produce superior sealing compared to

other materiais if another in vitro testing mode1 were to be used.

3) RESEARCH RATIONALE

Despite favorable in vitro biomechanical and microbiological results, there have been

no investigations into the biological safety of ZUT as an endodontic root-filling material.

This is an important consideration, since the usefùlness of a dental material may be limited

by its interactions with not only tissues of the oral cavity but the entire body. Effects may

range fiom being clinically insignificant to having considerably serious systemic results,

affecting both the patient and dental care providers (Smith and Williams, 1982). To assess

the hazard or safety of a new dental mateaiai to a patient, its biocompatibility must be

established.

Biological testing guidelines for dental materials have been put forth by the

American National Standards Institute in conjunction with the American Dental

Association (1982) and the Fédération Dentaire Internationale (1980). Three levels of

testing have been recommended for the biological evaluation of any dental material: 1)

initial tests, 2) secondary tests and 3) usage tests. Selected "initial" and "secondary" tests

should be employed to evaluate a new material pnor to the extensive "usage" tests. If the

results observed in a number of initial tests are obviously unfavorable (Le. not biologically

tolerated), the time and expense associated with tùrther initial screening and usage tests

can be precluded @DI, 1980).

3.1) STATEMENT OF THE PROBLEM

There have been no investigations to date reporîing the results of in viîro testing for

the biological safety of ZUT as a potential endodontic filling matenal.

3.2) OBJECTIVES

The objectives of this investigation are to characterize the hemolytic and cytotoxic

potential of ZUT, its GIC component (~etac-Cem@), and compare them to the commercial

endodontic sealers ~ e t a c @ - ~ n d o and AH 26@ (formulations both with and wit hout silver).

3.3) HYPOTHESIS

There are no natistically significant differences in the acute in vitro hemolytic

activity (using the Percent Hemolysis Test) and cytotoxicity (by the Millipore Filter

Method) of ZUT 0.2% (wt/wt ceramic component) in cornparison with the commercial

endodontic sealers.

4) METHODS AND MATERIALS

4.1) Rationale and operational definitions

According to Williams (1990), biocompatibility refers to "the ability of a material

to perform with an appropriate host response, in a specific application." ZUT is being

developed as a root filling matenal, potentially in direct contact with blood, epithelium,

connective tissue and inflammatory cells of the penradicular tissues. It is appropriate then,

to have chosen two tests to examine the interaction of ZUT with fiesh, whole blood and

human epithelial cells using the Percent Hemolysis and the Millipore Filter cytotoxicity

tests, respectively. These are accepted initial (screening) tests for the biological evaluation

of endodontic filling matenals put forth by the Arnerican National Standards Institute in

conjunction with the American Dental Association (ANSVADA, 1982) and the Fédération

Dentaire Internationale @DI, 1980). These guidelines state that the time and expense

associated with furiher "secondary" and "usage7' tests can be precluded if the results of

initial tests are obviously unfavorable (Le. significantly hemolytic and/or cytotoxic).

Assumptions

In this investigation it was assumed that in vitro studies of hemolysis and

cytotoxicity are both accurate and relevant rneasures of endodontic sealer biocompatibility.

Ideally, in vitro testing of biomaterials should match the ce11 populations to the typical

implant site (Oliva et al., 1996). Many cell lines have been used in previous endodontic

research (Table 15) but most investigators (e-g. Leirskar & Helgeland, 1972; Spingberg,

1978; Wennberg et al., 1979) have found no differences in reaction between ce11 lines

when used to evaluate cytotoxicity of endodontic materials. Considering the close

proximity of root filling materials to the vascular periodontium and oral mucosa, the

assumption of testing relevance regarding the effects on erythrocytes and an epithelial

(human epithelial, HeLa) cell line seems justified.

Table 15. Ce11 lines used to evaluate the cytotoxicity of endodontic materials (adapted from Murphy, 1988)

Guinea-pig leucocytes Human skin fibroblasts Mouse skin fibroblasts

Hamster kidney fibroblasts Human embryonic lung epithelial cells

Human HeLa epithelial cells Bovine pulp fibroblasts Human pulp fibroblasts

Human lymphoblasts Human lung fibroblasts

Human erythrocytes Human skin epithelial cells Human oral epithelial cells

Human lymphocytes and monocytes

The HeLa ce11 line (ATCC CCL-2) onginates fiom adenocarcinorna cells taken

during a cervical biopsy of an African-Amencan female human donor (Henrietta Lacks) at

the Johns Hopkins University Hospital in 195 1. This represents the first human epithelial

cancer ce11 line to be established in vitro and is recommended for use in the ANSUADA

(1982) and FDI (1980) testing guidelines in evaluating the biological safety of endodontic

root filling materials. By using this widely available ce11 line, it was hoped that uniform

cell behaviour was maintained, enabling comparison of these results with other

researchers.

The Millipore Filter test of cytotoxicity provides a means of comparing the

cytotoxicity of different endodontic sealers. This assay is based on the premise that the

enzyme act ivity of succinate dehydrogenase (SDH) is being measured, indicating the

presence of actively metabolizing ce1ls. The absence of its activity is suggests ce11

damage. Succinate dehydrogenase is an oxidative enzyme that facilitates the transfer of

hydrogen ions fiom succinic acid within the cytochrome system of mitochondria (Barka

and Anderson, 1963). The detection agent, nitroblue tetrazolium (NBT), is a tetrazolium

salt included in the incubation medium buffered at a neutral pH by phosphate. Normally

only slightly permeable to NBT, the inner mitochondrial membrane becomes more

permeable in the presence of the phosphate buffer. When succinic dehydrogenase oxidizes

succinate, the resulting hydrogen ion in turn reduces NBT to fom a blue formazan (Tyas,

1988). This insoluble product precipitates at the site of the dehydrogenase reaction.

resulting in a blue staining pattern on millipore filters with an adherent ce11 monolayer. An

advantage of this cytotoxicity assay is that the stained filters can be maintained as

permanent records of the test results (Hensten-Pettersen, 1988). The detection of SDH

using tetrazolium salts is accepted as a reliable indicator of active cellular metabolism

(Barka and Anderson, 1963).

It is also assumed that there is validity in comparing the cytotoxic and hemolytic

characteristics of ZUT 0.2% to the other dental materials. ~e tac@-~ndo and AH 26@ are

commercially available and have established biocompatibility data, so the comparative

cytotoxic and hemolpic properties detemined in this study are assumed to have both

clinical relevance and validity.

Limitations

Numerous differences exist when comparing in vitro and in vivo systems regarding

to the biological safety of materials. Cell behaviour may differ because in vitro specimens

are grown in isolation on a two-dimensional substrate (e.g. the miîlipore filtedagar

system), dissociated fiom cells they would normally interact with in vivo. Proliferation of

a single ce11 line (e-g. HeLa cells) represents a population of cells whose heterotypic

interactions with different ce11 types have been, for the most part, lost. Cellular metabolism

in vipo may be altered due to the absence of endocrine and nervous homeostatic

regulation. Although differences between the two systems are undeniable, in vitro testing

remains a valuable initial screening tool if the inherent limits of this testing environment

are recognized (Freshney, 1 987).

The preparation of test materiai is one of several difficulties encountered when

detennining cytotoxicity in vitro (Ciapetti et al., 1996). One limitation of the Percent

Hemolysis test recognized during the design of this study is the use of 5.0 grams test

material per measured sample, as suggested in the ANSYADA (1982) and FDI (1980)

guidelines. Five grams of ZUT 0.2%, for example, would require considerably large

amounts of powder and liquid components to be thoroughly rnixed and placed into ~eflon'

(Du Pont Co., Wilmington, DA) molds before setting. Due to the fast setting reaction of

the GICs, 5.0 gram samples represented an impracticably large amount to manipulate. The

ANSYADA (1982) guidelines state that it may be necessas, to reduce sample mass to as

little as 0.5 grams. After several preliminary experiments (see Appendices 1 to 5) a

smaller sample mass of approximately 1 gram test materiai was show to produce

consistent results and was used in the main hemolytic investigation. By using a reduced

test sample mass, but maintaining the equivalent blood solution vo1ume:test sampte mass

ratio (as per ANSYADA protocol, 1982), an adequate volume of supernatant was collected

for spectrop hotometric analy sis.

Unlike the Percent Hemoiysis test, the Millipore Fiiter cytotoxicity test enables the

examination of sealer samples allowed to set for variable periods of time fkom mixing

(including freshly mixed matenals). The surface area and volume of the material samples

exposed to the millipore filter (with the adherent HeLa ce11 monolayer) was standardized

using glass rings to contain the sealer samples. Within the scope of this examination, only

sealer material at the freshly rnixed stage and at various tirne periods up to 24 hours from

mixing were tested to determine cytotoxicity.

Delimitations

Consideration was given to determine the boundaries of the problem area in which

the investigation was confined. Only ZUT with 0.2% 2eomico> concentration (wt./wt.

cerarnic component) was evaluated in the proposed study. Dunng hemolysis testing, only

the percent hemolysis of test matenals that have been allowed to set for a period of 48

hours fiom mixing were investigated. A kinetic study of the hemolytic effects of the

sealers on percent hemolysis over time, for example, was not carried out.

The Millipore Filter test provided an indication of acute in vitro cytotoxicity of

sealer materials at different time penods up to 24 hours fkom mixing. Biocompatibility

over an extended time frame can be investigated utilizing the suggested Secondary Tests as

outlined by the ANSVADA testing guidelines (1982). The Bone Implant Test, for

exampie, provides an indication of tissue response to test materials after contact for a

period of six months. Evaluation of the biological safety of the test materials over an

extended time period of exposure, however, was beyond the scope of this investigation.

4.2) Selection of test materials

The proposed experiments teaed whether five endodontic sealers (independent

variables) have the same relative percent hemolysis and cytotoxic effects (dependent

variables) within the proposed testing systems. Three sealers have established

biocompatibil ity and are commerciall y available (i.e. ~e tac@-~ndo and AH 26@ wit h and

without silver formulations) while the two other sealers are in their experimental phase of

development (i.e. KT-308 and ZUT 0.2%).

Since KT-308 is currently in its experimental phase of testing, there may be

inconsistencies in both chemical composition and physical make-up between manufactured

batches. For this reason, and also due to the fact that potentially large quantities of GIC

would be required for the main investigations, ~etac-cema was substituted for KT-308 as

the GIC component of the ZUT formulation to be tested. ~etac-ceme is commercially

available as a restorative GIC product (not an endodontic sealer) but batches of this

material shouid be more consistent in composition than KT-308. It was a logical choice to

test ~etac-cema by itself (without any zeolite additive) to determine if the addition of

2eomica had an effect on its hemolytic and cytotoxic properties.

~e tac@-~ndo is commerciall y available and its clinical acceptability as a root canal

filling material has been documented (Kolokuris et al., 1996; Beltes et al., 1997). This GIC

sealer was well-tolerated when in contact with baby hamster kidney fibroblasts in vitro

(Beltes et al., 1997) and when implanted subcutaneously in Wister-Furth rats complete

healing was observed d e r 120 days (Kolokuris et al., 1996).

AH 26@ was selected as a test rnatenal because it represents a widely used,

commercially available endodontic sealer. This epoxy resin-based sealer is reporied to be

highly cytotoxic in vitro when fieshly mixed but inert d e r about twelve weeks (Eriksen et

al., 1988). Matsumoto and others (1989) found that fieshly mixed AH 26* altogether

stopped DNA synthesis of rat dental pulp cells in vitro and hypothesized that the

accelerator component (hexamethylene tetramine, HMT) was the cause of this cytotoxic

effect. Sphgberg and associates (1993) have since attributed the cytotoxicity of AH 26@

to the production of formaldehyde as a by-product of the HMT-catalyzed polymenzation

process.

4.3) Experimental methods

4.3.1) Percent Hemolysis Test

The hemolysis test evaluates the acute in vitro hemolytic activity of a material

intended for prolonged contact with bone and sofi tissue (ANSVADA, 1982) and can be

regarded as a cytotoxicity assay. The Percent Hemolysis test originates fiom the testing of

materials used for patients undergoing hemodialysis when it was observed that some

tubing and blood containers caused erythrocytes to rupture (Hensten-Pettersen, 1988). In

this particular assay, the test material is incubated in saline for a 90-minute period, with

rabbit blood present for the last 60 minutes. Both the physical surface of the test matenal

(affecting adherence and activation of the plasma protein systems and cellular components)

and its soluble, leachable components contnbute to the hemolytic activity examined by this

assay (Dillingham et al., 1975).

Since the concentration of a toxic material to produce hemolytic activity is

generally an order of magnitude higher than that required to produce a response in tissue

culture, hemolytic activity is considered an important indicator o f leachable toxic

components @DI, 1980). Hemolytic activity has been highly correlated with both tissue

culture response (Dillingham et al., 1975) and i n vivo acute toxicity (FDI, 1980). Tissue

culture and hemolysis in vitro tests are reported to provide the most information regarding

acute toxicity and are also the most sensitive to variables in the test material formulation

(Dillingham et al., 1975). Unlike the tissue culture system, the hemolysis assay system is

relatively unaffected by secondary time-dependent processes since there is: 1) a low

oxidative metabolism in erythrocytes; 2) an absence of metabolic act ivity associated wit h

growth and reproduction; and 3) the assay time for the tissue culture system is much longer

(e.g. for test material-ce11 contact and tetrazolium staining for SDH activity) than that

required for the hemolysis assay (Dillingham et al., 1983).

The following protocol is based upon that outlined in the ANSWADA (1982) and

FDI (1 980) testing guidelines for the evaluation of the biological safety of endodontic root

filling materials. Through a series o f preliminary Percent Hemolysis tests (Appendices 1

to 7), modifications to the original testing protocol were implemented (sections 4.4 and

4.4.1 ) to ensure reliable and reproducible results.

Materials

centrifige (CU-5000 LEC Centrifbge/Damon; Needham, MA) 37 OC water bath (Fisher-Scientific: Pittsburgh, PA) spectrophotometer (Spectronic 601, Bausch & Lomb Co.; Buffalo, NY) test tubes (16 mm x 100 mm; disposable, borosilicate coated; VWR brand, Mississauga,

ON) test tube silicone caps (ma brand, Mississauga, ON)

semi-micor cuvettes (disposable, polystyrene; 1.5 mL volume; DiaMed Lab Supplies Inc., Mississauga, ON)

rabbit blood (fiesh, whole, oxalated 2% w/v potassium oxalate in saline; equivalent to 1 mL/2O mL of rabbit blood); approximately 20 mL bjood required (Charles River Canada, St. -Constantine, Quebec)

ZUT (0.2% 2eomicm wlw glass ionorner, Shinanen Zeornic Co., Ltd., Aichi-Ken, Japan, ) ~etac-cemm (ESPE Arnerica, Inc., Nomstown, PA) ICetacm-~ndo Apiicap (ESPE Arnerica, Inc., Nomstown, PA) AH 26@ (formulations both with silver and silver-free; Dentsply DeTrey GrnbH,

Konst anz, Geman y)

~ u l ~ ~ e n t " 1 cc disposable syringes (Watertown, MA) serological pipettes (disposable; 1, 5, 10, 20 cc sizes; alc con@ brand, Becton Dickinson

Co., Franklin Lakes, NJ) phosphate buffered saline (PBS) solution (Dulbecco's PBS without calcium, without

magnesium; 137 m M NaCl, 2.7 mM potassium chlonde, 10 m . phosphate buffer, pH 7.4; Sigma Aldrich Co., St. Louis, MO)

0.1% sodium carbonate solution (Sigma Aldrich Co., St. Louis, MO) dental mixing spatula mixing pad disposable gloves (Anse11 Perry Inc., Massillon, Ohio) disposable masks (secure-~ard@ brand; Arnerican Threshold Healthcare, Enka, NC)

Preparation of rabbit blood

The anticoagulated rabbit blood was diluted with PBS solution. This was

satisfactorily diluted when a volume of 0.2 rnL of diluted blood was hemolyzed in 10 mL

of 0.1% sodium carbonate solution and produced a spectrophotometric reading of 0.95 k

0.5 optical density (OD) at 545 nrn wavelength (A).

Preparation of test materials

The ZUT test rnaterial was made by adding ~eomic@ to the glass component of

~etac-cerna to a total weightheight ratio of 0.2% under aseptic conditions (e.g. 0.0668 g

~eomic@ into one bottle of ~ e t a c - ~ e m @ containing 33.4 g glass powder). The bottle

containing this mixture was shaken vigorously and stored at room temperature out of direct

sunlight until required for testing.

Using a laminar flow hood, the liquid and powder cornponents of ZUT material

were prepared on a mixing pad using a dental spatula to a thick, creamy consistency

(approximately two drops to one scoop of powder; as per manufacturer's recommendations

for ~etac-cema material). This was then quickly spatulated into the pulp~enta syringes,

and allowed to set in an incubator (at 37 OC, 5% CO2, 100% r.h.) for a penod of 24 hours.

Pieces of the test material were sectioned from the resulting rod (using a sterile razor

blade), weighed, and divided into test sarnple batches of 0.5 gram (one tenth of the

recommended test material mass in the ANSVADA guidelines, 1982). The same

procedures were repeated for the other test materials.

Methodology

Sealer materials (0.5 g each) were placed in a test tube into which 10 mL PBS

solution was added, covered with test tube caps, and then equilibrated in a 37 OC water

bath for 30 minutes. Diluted rabbit blood solution (0.2 rnL) was added to each tube, mixed

by gentle inversion, and incubated in the water bath for another 60 minutes.

A positive control sample (representing 100 % hemolysis) was made by adding 0.2

mL of the diluted blood solution to 10 rnL 0.1% sodium carbonate solution and mixed

gently. Adding 0.2 rnL of the diluted blood to 10 rnL of PBS solution provided the

negative control sample (an estimate of spontaneous lysis). Both the positive and negative

control tubes were prepared and incubated in the same manner as the tubes containing test

materials.

When the 60-minute incubation period elapsed, the tubes were centrifuged at 750 x

g (adequate force to pellet the erythrocytes) for ten minutes and the resulting supernatant

was transferred to spectrophotometnc cuvettes. Optical density (O.D.) readings were

determined at 545 nm ?i and recorded to calculate the percent hemolysis using the

following formula:

4.3.2) The necessity of preliminary testing

As suggested by Darby and Bowen (1980), valuable insights into the validity and

reliability of the planned study, as well as any inherent design flaws can be elucidated from

initial experiments and adjustments for the main experiment made accordingl y.

Preliminary Percent Hemolysis (Appendices 1 to 7) and Millipore Filter (Appendix 8) tests

were conducted to determine any shortcomings in the feasibility and practicality of the

proposed investigations.

4.3.3) Modifications to the Percent Hemolysis Test protocol

The effect of test material surface area

The results of a preliminary percent hemolysis test using four endodontic sealers

(summarized in Appendix 1) suggest that there were no statistically significant differences

in percent hemolysis between matenal groups (Kruskall-Wallis test; p = 0.433, a = 0.05).

The large variances within each sealer group, however, indicated that modifications should

be made to increase the precision of the hemolysis assay. Since surface area of the

material has a direct effect on the release of hemolytic products (Dillingham et al., 1979,

it was decided to maintain a uniform surface area for the samples instead of standardizing

only by mass.

ïeflon" molds were processed to create cylinders of test material (7 mm diameter x

12 mm long; approximate mass 1 gram) with a consistent surface area (340.69 mmz). The

materials were allowed to set for 48 hours (at 37 C; 100% r.h.) and the experiment was

repeated. The results of this preliminary study (contained in Appendix 2) show an

unexpected negative mean percent hemolytic effect of AH 26@ and ~etac-cem? The

blood cells were observed to agglutinate during incubation of the test materials and blood

solution. This hemagglutination may have protected them fiom the potential hemolytic

effects of the test materials resulting in the negative hemolytic findings. Further changes

to the assay methodology were advised after collaboration with an expert in the field of in

vitro hemolytic testing of medical biomaterials (George Adams, Ph.D., Innovations

Foundation, University of Toronto).

Potassium oxalate as an anticoagulation agent

Potassium oxalate is the suggested blood anticoagulant in the ANSVADA

Recommended Standard Practices for the Biological Evaluation of Dental Materials

(1982), however, due to the observed blood coagulation and negative percent hemolytic

results, this agent was determined to be inadequate as an anticoagulant at the dilution

levels useci in this study. Currently, there is no consensus on the ideal in vitro blood

anticoagulation agent (Meuller et al., 1993), but it was decided to use

ethylenediaminetetraacetic acid (EDTA) because of the higher calcium- and magnesium-

binding afinity for this chelating agent and its resultant decrease in agglutination.

The importance of the blood-saline voiume:test material mass ratio

A preliminary percent hemolysis study using blood anticoagulated with EDTA (2

mg EDTNmL total volume, as specified by Freshney, 1988) resulted in considerably less

negative hemolysis (Appendix 3). The presence of negative values, however, suggests an

unlikely protective effect of the red blood cells by the materials against hemolysis. The

large variances also suggest that the modifications to the original protocol did not produce

the desired increase in assay precision. Further consultation with Dr. Adams resulted in

attempts to maintain a consistent blood-saline solution volume:test material mass ratio.

Rather than using the same volume of blood solution for each test sample, the volume was

instead adjusted for each test sample mass. The amount of blood exposed to the test

material, based on its mass, was kept equivalent to that specified in the ANSUADA

protocol (1982) for testing matenal samples with a 5.0 gram mass (2 rnL blood-saline

solutiodgram test material). For example, 1 .O gram of test material was incubated in PBS

solution containing diluted blood solution at a concentration of 2% v/v (i.e. 1-96 mL PBS +

0.04 mL blood solution), producing sufficient supernatant (minimum volume

approximately 1.2 mL) for measurement of optical density using the spectrophotometer.

By adhering to the equivalent blood solution vo1ume:test material mass ratio, using

EDTA as an anticoagulant, and also maintaining a consistent surface area among test

matenal samples, it was hoped to increase the precision of this assay. A Percent

Hemolysis test utilizing these methodological modifications was performed and resulted in

mean hemolytic values within the expected range (40%) with no significant differences in

hemolysis among the test materials (one-way ANOVA with Duncan's painvise t-tests; p =

0.25 14) (Appendix 4). With an acceptable Percent Hem01 ysis testing methodology

developed, matenal testing was conducted using cylinders of materials and repeated to

evaluate the reproducibility of this assay (Appendix 5).

Determination of inherent error in this testing system

AIthough the methodology of the hemolytic testing appeared to now produce

consistent and reproducible results, the standard errors still accounted for a large

proportion of the mean percent hemolpic recordings. The ermr range produced could be

inherent in this hemolytic testing system and remain constant (regardless of the amount of

hemolysis) or it could possibly be dependent upon the overall amount of hemolysis

produced. To determine if the standard errors increased in proportion to the hemolysis

produced, an attempt was made to increase the overall hemolysis by increasing the surface

area of the material in contact with the blood solution. The test material surface area:mass

ratio was increased by a factor of 2.5 by creating smaller disks (7 mm diameter x 3 mm

height) formed in a ~e f lon@ mold. The hemolysis test (using 6 diskdtest sample; surface

area = 857.22 mm2) was perforrned in the same manner, as described in Figure 4 below,

and repeated to ver ie reproducibility of the results (shown in Appendix 6).

I - Positive hkolytic control

Centrifugation (750 x g)

Supernatant OD,. ,,, 1 readings

Figure 4. Su- of the Percent Hemolysis Tesq rnethodology

Statist ical tests (one-way ANOVA) were used to determine if test material surface

area had an effect on percent hemolysis produced. The mean values of the Percent

Hemolysis assays are presented in the "Results" section of this investigation.

Possible effect of solu ble p d u c t s on spectrophotornetric readings

The soluble products leached from the test materials, following incubation Ui the

hot water bath, may have had an effect on the spectrophotornetric absorbance readings. If

so, the increased release of these products fiom the diAs of test materials (due to an

increased surface area) could result in misinterpretation as increased hemolysis produced

after the blood solution was added. In order to test the effect of products leached into

solution foiiowing incubation, disks of ~etac-cerna and AH 26@ (with silver) were pkced

in PBS solution (using the same PBS vo1ume:test mass ratio used in the Percent Hemolysis

testing protocol) and incubated in a hot water bath (at 37 OC) for 90 minutes. Following

centrifbgation (750 x g) for 10 minutes, the supernatants were measured for optical density

(545 nm h) and compared to ccntrols (PBS solution). This experiment was repeated and

the results analyzed using a one-way ANOVA (Appendix 7).

4.3.4) Millipore Filter Test of Cytotoxicity

This method is an assay of cytotoxicity based on indirect cell-material contact

where a monolayer of target (HeLa) cells is grown on one side of a molecular filter and the

test specimen is placed in contact with the other side (Wennberg, 1988). Since SDH is an

essential enzyme involved in the basal metabolic activities of cells, the filter is stained for

the detection of SDH activity as an indicator of active metabolism (Barka and Anderson,

1963).

This test was originally developed to serve as a simple method for the toxicity

screening of liquid, setting, and solid materials (Wennberg et al., 1979). Unlike the direct

contact inhibition tests, the potential discriminating effects of agar on certain soluble

components of the test material are eliminated, as the nutrient-agar medium used in the

Millipore Filter test serves only to support and nourish the ce11 monolayer during the

contact period with the test materials. Hematoxylin and eosin staining has s h o w the

intimate proximity of ce11 processes extending through the millipore filter (125 p

thickness) and ont0 the test matenals when observed under light microscopy (Wennberg,

1988). Hurnan epithelial (HeLa) cells were found to grow better on 0.45 pm pore size

filters compared to 3.0 and 8.0 pm pore sizes (Wennberg et al., 1979), thus the former

fiber pore size is the standard for this test. Based upon these findings, it can be assured

that the HeLa cells will be in very close proximity, albeit via indirect contact, and provide

an indication of the test materials' cytotoxic potential.

The following testing protocol is that outlined in the FDI testing guideline for the

evaluat ion of the biological safety of endodontic root filling materials (1 980). Through a

series of pilot studies (Appendix 8), modifications to the original testing protocol were

implemented (section 4.4.2) to ensure reliable and reproducible results.

Materials

laminar flow hood (Forma Scientific, Inc; Mariette, Ohio) mass scale (Sartorius, Gottingen, Germany) incubator (37 OC, 5% COz in air, 90 * 10Y0 r.h; Sanyo CO2 mode1 MCO- 1 7 4 Japan) hot water bath (40 OC; Fisher-Scientific, Pittsburgh, PA) millipore filter disks (47 mm diameter, 0.45 pm pore size, white surfactant free; cat. no.

HATF04700; Millipore Corporation, Bedford, MA) tissue culture petri dishes (polystyrene, 50 mm diameter x 10 mm size; al con" brand,

Becton Dickinson Co., Franklin Lakes, NJ) serological pipettes (polystyrene disposable; 1, 5, 10, and 25 cc volumes; al con" brand,

Becton Dickinson Co., Franklin Lakes, NJ) Pasteur pipettes (VWR@ brand; disposable, 22.9 cm length; Mississauga, ON) centrifuge tubes (15 and 50 mL sizes, polypropylene; alc con" brand, Becton Dickinson

Co., Franklin Lakes, NJ) cryovials (1.5 rnL volume; BarnsteadThermolyne Co., Dubuque, IA) 0.25% trypsin - 0.03% (w/v) EDTA solution (Sigma Aldrich Co., St. Louis, MO) dimethyl sulfoxide (DMSO; C2&0S; Sigma Aldrich Co., St. Louis, MO) trypan blue solution (0.4%; Sigma Aldrich Co., St. Louis, MO) ELISA plate (Dynex Technologies, Middlesex, U.K.) micropipeîtor (20pL; VWRabrand, Mississauga, ON ) hemocytometer (0.4 Mm; Fisher Scientific, Nepean, ON) tissue culture flasks (T75) (250 mL, polystyrene, order number 353 11 1; al con" brand,

Becton Dickinson Co., Franklin Lakes, NJ) phosphate buffered saline (PBS) solution (Dulbecco's PBS without calcium, without

magnesium; 137 m M NaCI, 2.7 mM potassium chloride, 10 rnM phosphate buffer, pH 7.4; Sigma Aldrich Co., St. Louis, MO)

distilled water

ZUT (0.2% 2eornica w/w g l a s ionorner, Shinanen Zeomic Co., Ltd., Aichi-Ken, Japan) ~etac-cerna (ESPE America, Inc., Nomstown, PA) ~ e t a c @ - ~ n d o Aplicap (ESPE America, Inc., Nomstown, PA)

AH 26@ (formulations both with silver and silver-fiee; Dentsply DeTrey GmbH, Konstanz, Germany)

glass rings (inner diameter 40 mm, outer diameter 47 mm, height 5 mm; autoclaved; Chemical Engineering Glassworks Laboratory, University of Toronto)

glass rings (inner diameter 7 mm, height 5 mm; autoclaved; Chemical Engineering Glassworks Laboratory, University of Toronto)

~ o n o j e t ~ 3 cc endodontic syringes (23 gauge; Sherwood Medicai Co., St. Louis, MO) dental mixing spatula mixing pad

Ce11 Iine: human epithelial cells (HeLa; ATCC CCL-2; American Type Culture Collection, Manassas, VA)

Growth medium (500 mL total volume): 3 89.4 mL Eagle' s a-MEM (minimum essential media) 5 mL HEPES buffer (= 10 mM) 5.6 rnL sodium bicarbonate buffer (= 10 rnM) 50 mL antibiotics (10x concentration) 50 mL x 10% fetal calf serum (FCS) (al1 Sigma Aldrich Co., St. Louis, MO)

10x Antibiotic solution (200 mL total volume): 20 mL x 10% fetal calf serum 10 000 U penicillin (50 U h L ) 10 000 pg gentimycin (50 pg/rnL) 50 pg arnphotericin B (0.25 pg/mL) (al1 Sigma Aldrich Co., St. Louis, MO)

Agar medium (100 mL total provides enough to make 20 petri dishes; 5 mL/dish): 73 mL Eagle's a-MEM 1.5 g ~ a c t o ~ ~ Agar (Difco" brand, Becton Dickinson & Co., Sparks, MD) 1 O mL antibiotics (1 0x concentration) 1 mL HEPES (=IO mM = 1 mL/100 mL medium) 1.12 m . sodium bicarbonate solution (= 10 mM) 1 5 mL x IO% fetal calf serum (al1 Sigma Aldrich Co., St. Louis, MO unless othenvise stated)

Sodium succinate solution (to make 100 mL of 0.06 M solution): succinic acid (butanedioic acid; F.W. 270.1) disodium salt hexahydrate (Sigma

Aldrich Co., St. Louis, MO) If: 270.1 gram succinic acid into 1 L distilled, filtered water = 1 M solution Then: 16.2 gram succinic acid into 1 L distilled, filtered water = 0.06 M solution (1.62 g succinic acid into 100 mL = 0.06 M) pH set to 7.0; stored in a sterile bottle covered with foi1 at 4 'C

Nitro blue tetrazolium chloride monohydrate (0.2% solution) 100 mg nitro blue tetrazolium (Aldrich Chemical Co., Milwaukee, WI) into 50 mL distilled, filtered water. pH set to 7.0-7.4; stored in a sterile bottle covered with foi1 at 4 OC).

Note: sodium succinate and Nitro BT solutions may be stored at 2 - 4 OC for months parka and Anderson, 1963).

Incubating medium (to stain filters for SDH detection): 20 rnL x 0.06 M sodium succinate solution 50 rnL x 0.2% Nitro Blue tetrazoliurn chloride monohydrate solution 20 mL phosphate buffered saline (PBS) solution @ulbecco's PBS without calcium,

without magnesium; 137 mM NaCl, 2.7 mM potassium chloride, 10 mM phosphate bufler, pH 7.4; Sigma Aldrich Co., St. Louis, MO)

10 mL lactated Ringer's solution (273 mOsm/L, pH 6.7; Abbott Laboratones Ltd., St. -Laurent, Quebec)

Culturing of the HeLa ce11 line:

Al1 procedures were performed using aseptic technique under laminar flow hood.

HeLa cells were received fiom the ATCC in the form of ce11 suspensions which were

seeded into two T75 culture flasks containing 12 mL growth medium, then grown in an

incubator (37 OC, 5% CO2 in air, 90% * 10% r.h.). Growth media was changed every 2 - 3

days.

When cells grew to confluence (approximately every 5 days), they were "split" to

seed 10 x T75 flasks to produce an adequate number of cells for long-term storage in liquid

nitrogen.

The procedures used to seed a new passage of HeLa cells are as follows:

Culture flasks were observed under the light microscope to confinn their confluent

growth.

Growth media was aspirated fiom the culture flasks using a Pasteur pipette, taking

care not to aspirate the confluent monolayer of HeLa cells attached to the bottom.

Approximately 3 rnL of PBS solution was used to briefly rinse any media and

debris (e.g. dead cells) fiom the HeLa cells adhered to the flask. This was repeated

for a total of three rinses per flask.

Two mL of 0.25% trypsin - 0.03% EDTA solution (w/v) (warmed to 37 OC) was

pipetted into each of the culture flasks, which were then transferred to an incubator

for 3-4 minutes. The HeLa cells were enzymatically cleaved fkom the polystyrene

flask and appeared as opaque, white clusters. Under a light microscope, the cells

now appeared as free-floating spheres.

The trypsinization process was stopped by the addition of 4 mL growth medium

(containing 10Y0 FCS). Cell damage can occur if the ce11 culture is exposed to the

trypsin-EDTA solution longer than the recommended time.

Ce11 suspensions were then transferred into centrifuge tubes (50 mL volume sizes)

and centrifuged at 1 100 r.p.m. for 12 minutes.

The resulting supernatant was suctioned off of the pellet of cells, which were then

resuspended into 12 mL of growth media.

A hemocytometer was used to estimate the ce11 concentration (see below) and

additional growth medium was added to produce a final ce11 concentration of 7.5 x

10' cells/mL suspension. One mL of this suspension was sufficient to seed one

T75 culture flask containing 12 mL growth media (with the recommended 10 000

cells/cmz) when a new ce11 line was started or ongoing cultures required. These

were maintained until ce11 confluence was reached, then trypsinized and used to

seed anot her ce1 f passage as before.

One rnL of ce11 suspension was then placed in a cryoviaf and

dimethylmethylsulfoxide @MSO, a cryoprotectant) was added to a final

concentration of 5% v/v.

Five cryovials of HeLa cells were produced and stored in gradually cooler

conditions as follows:

a) 30 minutes in a refrigerator at 4°C.

b) 2 hours (minimum) storage at -70 OC.

c) Long-term storage in liquid nitrogen (-196 OC) using the facilities at the

MRC Periodontal Research Group ( 2 " floor, Fitzgerald Building; contact

Mr. C heung Lo; storage boxes #96-# 100 label led "HeLd28 Oct. 991David

Thom")

The HeLa ce11 line could be maintained indefinitely but if it was to be discontinued

(e-g. due to possible contamination of the cell cultures), the cryovial suspensions

stored in liquid nitrogen could be thawed and used to seed a new T75 culture flask

containing 12 m . of growth medium.

Determination of cell concentration:

When culture flasks containing a confluent layer of HeLa cells were trypsinized

and then resuspended following centrifugation (see protocol above), it was necessary to

determine the ce11 concentration:

1) 20 pL cell suspension was mixed with 20 pL trypan blue dye in the well of an

ELISA plate.

2) This mixture was pipetted under a glass cover slip on a hemocytometer.

3) Using a light microscope, the average number of cells counted for the gndded

quadrants was calculated. Ce11 concentration was determined as follows:

x = average number of cells in each quadrant of the hemocytometer grid 2 = dilution factor (cell suspension + dye solution) 104 = conversion factor of hemocytometer

so: x (2) x (10') = number of cells/mL suspension

Preparation of the agar medium

The a-MEM solution and ~ a c t o ~ ~ Agar were placed into a glass bottle (250 mL

volume) and, with the top lwsely twisted on, sterilized in an autoclave. Immediately afier

stenlization, the bottle was cooled to 50 "C in a hot water bath. Under a lamina flow hood

and using aseptic technique, the remaining agar medium constituents were pipetted into the

bottle followed by gentle mixing. Into each empty tissue culture dishes (50 mm diameter),

5 mL of agar medium was pipetted and allowed to solidi@ at room temperature. These

agar dishes were then packaged and stored at 4 OC for up to one month prior to use for the

Millipore Filter test.

Preparation of the millipore filter ce11 cultures

One millipore filter was placed into a tissue culture dish (50 mm diameter) and a

large diameter glass ring was then placed on top of the filter to prevent its movement in

liquid medium. Cells from stock culture were suspended in growth medium to a

concentration of 1.5 x 10' cells per mL. Six mL of this ce11 suspension was pipetted on top

of the filters. These were then placed in an incubator (at 37 OC; 5% C a ; 90 * 10% r. h.)

for a 24 hour period.

Preparation of test materials

Each sealer was mixed according to the manufacturer's instructions under aseptic

conditions using a laminar flow hood. The AH 26@ materials were placed into ~ono je t@

endodontic syringes then injected into the smaller diameter glass rings. The GIC matenals

were placed into the small diameter glass rings using a dental spatula after mixing. The

test samples were then allowed to set at 37 OC (90 1094 rh.) for 24 hours. This procedure

was repeated for materials to be tested at 1, 2, 3, and 6 hours after mixing. AH 26@

material to be tested 3 hours from mixing, for example, was mixed, placed in a ~onoje t"

syringe and transferred to an incubator for 3 hours. At this time, the AH 26@ material was

injected into the small diameter glass rings placed on top of the millipore filterlcell

culturdagar medium system. Freshly mixed materials were prepared in the same manner,

but the smaller diameter glass rings were first placed on top of the prepared millipore

filterlcell culture/agar medium system prior to loading the sealer.

Methodology

A millipore filter with an established HeLa cell monolayer was rinsed once in PBS

solution (pre-warmed to 37 OC) and placed cell-side down ont0 the agar surface. Five test

specimens were placed on top of each millipore filter. Ten specimens of each material, as

recommended by the FDI (1982), were teaed in this manner and again repeated. Control

specimens were millipore filters with an established ce11 monolayer but not in contact with

test specimens. A second control included filters without a cell layer but in contact with

test specimens. To determine the effect of test sarnple weight on the filter staining pattern,

glass rings containing dental wax served as test controls.

The culture dishes (containing the test materials contacting the filters and HeLa

cells) were then placed in an incubator for a 2 hour contact period (at 37 OC. humidified

atmosphere of 5% CO2 in air), d e r which time the test samples were discarded, the filters

gently removed from the agar layer, rinsed in PBS solution, and then incubated ovemight

for detection of SDH activity of the HeLa cells. After the staining procedure was

completed, the filters were rinsed in distilled water, allowed to air-dry, and then mounted

under a protective covering for assessrnent of the staining pattern.

Assessment of millipore filters:

The following sconng system and its interpretation, as recommended by the FDI

(1982), was used to describe the appearance of the test filters at the cell-material contact

areas:

O: no difference in staining intensity as compared to the rest of the ce11 layer 1: zone of reduced staining intensity, or an unstained zone, with a diameter = 7

mm (diameter of the test specimen) 2: an unstained zone 7-1 1 mm wide 3: an unstained zone 12 mm or wider

Interpretation of millipore filter scores:

The interpretation of relative cytotoxicity, based on the scoring system previously

mentioned, was as follows:

0: Non-cytotoxic response 1: Mild cytotoxic response 2: Moderate cytotoxic response 3: Marked cytotoxic response

4.3.5) Modifications to the Millipore Filter Test protocol

Effct of HeLa cell confluence on filter staining intensity

A pilot study was conducted using millipore filters (with no test materials) placed

in contact with a suspension of HeLa cells for a 24-hour penod, as suggested in the FDI

guidelines (1 980). The filter staining intensity after incubation ovemight in a tetrazolium

blue dye solution (an indication of metabolically active cells) was relatively weak

compared to that described in previous literature (Wennberg, 1980). It was decided to

conduct a histological examination of filters in contact with the HeLa ce11 suspension for

periods of 24, 48, and 72 hours to visualize the confluence of the resulting HeLa

monolayer. Presumably, a more confluent layer of viable cells will result in a darker and

more intense blue colour afler SDH staining procedures. When testing with the endodontic

sealers, a more distinct border between unstained and stained zones could be visualized,

enabling a more accurate recording and interpretation of cytotoxic staining patterns.

Using the method described by Wennberg (1988), filters were observed under light

microscope to have a sparse density of cells at the 24-hour period, becoming progressively

more confluent after a culture period of 72 hours (Appendix 10). A cross section of a 72-

hour ce11 culture on a millipore filter verified the presence of a confluent ce11 monolayer

adhenng to the filter surface (see Figure 5). Based upon these findings. it was decided to

incubate the millipore filter in the HeLa cell suspension for a 72-hour penod pnor to

cytotoxicity testing in an effort to produce optimal staining intensity.

wax control

HeLa monolayer

I miIlipore filter l n 2 hour contact te-oijum --A-- -Li-- dye

L hr. rrom mixïng

Figure 5. Surinnary of Millipore Filter Test of Cytotoxicity methodology

4.4) Data analysis

AU statistical tests and interpretations were perforrned with the invaluable

assistance of Dr. Gajanan Kuikami. The critical values of all statistical tests used (for both

the Percent Hemolysis and Miiiipore Filter assays) were set at the 5% significance level [a

(Ztailed) = 0.051. Descriptive data analysis (graphs, tables, charts, etc.) and statistical

techniques (measures of central tendency, dispersion, analysis of variances) for both assays

were perfomd using SAS 6.12 statistical software for Widows and Microsofi Excel97.

4.4.1) Percent Hemolysis Test

Summary statistics such as mean (* S.E.M.) for the resulting percent hemolysis

values were compared using a single-factor analysis of variance (ANOVA) to test the nul1

hypothesis: there is no difference in mean percent hemolysis values of the different

endodontic sealer materials. In the event of a significant F-ratio, a Duncan's painvise t-test

of means was employed to identify significant pairwise comparisons among the groups.

4.4.2) Millipore Filter Test

Summary statistics, univariate and multivariate analyses were conducted to detect

statistically significant differences in the diameter of unstained zones for each sealer type

at different time periods from mixing as well as comparing the different sealer materials.

A one-way analysis of variance (ANOVA) to test for differences in the diameter of

unstained zones was employed for each time period fiom mixing and a Duncan's painvise

t-test of means used to identi& significant painvise comparisons among groups.

4.5) Resources and environment

Hemolysis testing and al1 statistical analyses were performed at the University of

Toronto, Faculty of Dentistry Building. Cytotoxicity assays were conducted at the

University of Toronto Institute for Biomaterials and Biomedical Engineering. Both

facilities had the required safety standards authorizations, personnel with the necessary

expertise, and equipment needed to carry out the aforementioned protocols.

5) RESULTS

5.1) Percent Hemolysis Test results using cylinders of materials

As s h o w in Figure 6, there were no significant differences in the percent hemolysis

produced by cylinders of test materials (1-way ANOVA with Duncan's pairwise t-tests; p>

0.05). Cylinders of ~e t ac -~em" , AH 26' (formulations bot h with si lver and si lver-free).

Figu

~etac-cerna AH 26@ AH 26@ ZUT 0.2% (with silver) (silver- free)

re 6. Percent Hemolysis Test results using cylinders of test materials (n = 6; repeated twice; expressed as mean + S.E.M.; sample surface area was 340.69 mm2 and the sample weight to blood solution volume ratio was 0.50 g/rnL)

and ZUT 0.2% produced mean percent hemolysis values ranging from 5.56% to 7.75%.

Negative hemolytic values were not produced. Due to a limited supply of ~ e t a c - ~ n d o *

material, cylinder samples were not evaluated in this main experiment . Statistical analysis

of preliminary test results found no differences in the hemolytic acitivity of ~e tac -~ndo"

compared to the other GIC test materials, so its withdrawal fiom testing for practical

considerations was justified. A detailed statistical analyses of results are provided in

Appendix 8.

5.2) Percent Hemolysis Tests using disks of material

Using disks of test materials, there was no difference in percent hemolysis produced

by the test materials, with the exception of AH 26@ (with silver formulation) Vigure 7).

~ e t a c - ~ e m " ZUT 0.2% ~ e t a c @ - ~ n d o AH 26@ AH 26@ (silver-free) (with silver)

Figure 7. Percent hemolysis test results using disks of test materials [n=6; repeated twice (except AH 26" silver-fiee matenal, not repeated); expressed as mean a S.E.M.; 'denotes significance at p<0.0001; sample surface area was 857.22 mm2 and the sample weight to blood solution volume ratio was 0.5 g/mL]

~etac-~em@, ZUT 0.2%, ~ e t a c @ - ~ n d o and AH 26@ (silver-fiee) disks resulted in a similar

range of hemolysis (5.35 to 11.0%) as their cylinder counterparts. AH 26@ (with silver)

disks produced a mean percent hemolysis value of 33.1% (S.E.M. = 7-37), significantly

greater than al1 other disks and cylinders of test materials (1-way ANOVA with Duncan's

painvise t-tests; p<0.0001). The percent hemolysis produced by ~e tac@-~ndo and Ketac-

cerna disks was significantly different at a = IO?% but not at the 5% significance level

(two-tailed student t-test of means; p = 0.07). A detailed statistical analysis is included in

Appendix 8.

The possibility of non-hemolytic leachates fiom the test materials affecting optical

density readings (OD at 545 nm k) was considered. An experiment was performed in

which disks of AH 26@ (with silver) and ~etac-cemm were incubated in PBS solution

(containing no blood; in a hot water bath at 37 OC) at the same vo1ume:test material mass

ratio as in the main experiment. Compared to control samples of PBS, there were no

significant differences in OD readings for both test materials (Table 16; 1-way ANOVA

with Duncan's pairwise t-tests; p>0.3; Appendix 9).

Table 16. Optical density (OD) readings of test matenals (6 diskdtest sample) afier 90 minute incubation in PBS at 37 OC - experiment repeated twice

PBS Alone (no test material)

6 -0.026 0.0038

Sample size Mean OD (545 nm) Standard error of mean (SeEeMe)

AH 26@ (with silver) in PBS

6 -0.029 0.00 17

~ e t a c - ~ e m " in PBS

6 -0.03 1 0.001 1

5.3) Results of the Millipore Filter Test of cytotoxicity:

Unstained zones produced on the millipore filters after incubation in tetrazolium blue

dye solution identify areas of HeLa ce11 death. The diameter of the unstained zone reflects

the relative degree of cytotoxicity of the test material (Le. a larger unstained zone suggests

a greater cytotoxicity; end-point criteria is described in Section 4.3.4). The line graph in

Figure 8 shows clear trends in the unstained zone diameters produced by the test materials

from the freshly mixed stage up to 24 hours from mixing. Al1 freshly mixed sealers

produced a "moderate" cytotoxic response (unstained zone 7-1 1 mm diameter), with

~ e t a c @ - ~ n d o producing the most cytotoxic response (mean diameter = 11 -7 mm ; 1-way

ANOVA with Duncan's pairwise t-tests; p<O.0001). At the one hour time penod from

mixing and subsequent time periods tested t hereafler, the three GIC materials (lCetaca-

Endo, ~ e t a c - ~ e m @ , and ZUT 0.2%) produced no unstained zones on the millipore filters.

This "non-cytotoxicyy response at one hour from mixing was not exhibited by the

two AH 26@ sealer formulations. Instead these materials both elicited an ongoing

"moderate cytotoxic" response for up to 3 hours from mixing. At this time period, the

mean unstained zone diameters for AH 26& with silver (8.43 * 0.12 mm) and AH 26"

silver-free (7.63 I 0.2 mm) formulations were significantly more cytotoxic than the other

test materials (1-way ANOVA with Duncan's pairwise t-tests; p<O.0001). At 6 and 24

hours fiom rnixing, both AH 26@ formulations produced a "non-cytotoxic" response (no

unstained zone produced).

As expected, wax controls produced no changes in staining intensity on the

millipore filten (a "non-cytotoxic" response) when used to test the effect of weight alone.

Macroscopic examination of the wax control filters showed a ceIl monolayer uniformly

staining dark blue in colour (Appendix IO), consistent with the FDI Guidelines (1980). A

detailed statistical analysis of the Millipore Filter test results is included in Appendix 1 1 .

6) DISCUSSION

This investigation provided important information to characterize the cytotoxicity of

ZUT relative to commercial endodontic sealers and also identified factors that influence the

Percent Hemolysis and MiIlipore Filter tests.

ZUT as a potential endodontic sealer

Investigations into the biomechanical (Lalh et al., 1999), antimicrobial (Patel et ai.,

2000), and now, acute in vitro biocompatibility of ZUT (Figures 6 - 8) suggest this matenal

to possess characteristics equal or superior to those of other root filling materials currently

available. Based on existing studies, the ability of ZUT to prevent bacterial colonization of

obturated canals rernains in question (McDougall et al., 1999; Padachey el al., 2000).

Results of the previous ZUT leakage studies, however, must be interpreted with caution

because a lack of correlation ofîen exists between the sealing effectiveness of a test material

and its behaviour in commonly used leakage tests (Barthel et al., 1999; Pomme1 et al.,

2001).

The two in vitro screening tests used in this investigation for the biological evaluation

of ZUT 0.2% suggest this material to possess cytotoxic and hemolytic properties that are

better or similar to those of ~ e t a c @ - ~ n d o (Figure 8), a commercially available endodontic

sealer. Compared to the established AH 26@ (with silver and silver-free) sealer

formulations, ZUT 0.2% had a reduced hemolytic potential and a shorter duration of

cytotoxicity when setting. These favorable results encourage the further developrnent of

ZUT as a potential endodontic root filling material.

Hemolytic properties of the test materials

There were no significant differences (Lway ANOVA; p>0.05) in the percent

hemolysis produced by the ZUT 0.2% and ~ e t a c - ~ e m @ test sarnples. This indicates that

the addition of 0.2% ~eomic" (wt./wt. GIC ceramic) in the formulation of ZUT did not

result in different hemolytic characteristics than those of the GIC component of ZUT.

AH 26" (with silver) produced significantly more hemolysis when tested in the disk

form compared to the cylinder form (see Figures 6 and 7). Both the physical surface and

the soluble teachable components of the test rnaterials contribute to the overall hemolysis

detected in the Percent Hernolysis assay (Hensten-Pettersen, 1988). In the disk fom, the

surface area was increased over that of the cylinder form by a factor of nearly 2.5. This

change is believed to be in part responsible for the increased percent hemolysis f33.1 (h

7.37 S.E.M.) versus 5.58 (* 1.98 S.E.M.) mean percent hemolysis, respectively]. An

increased surface area may have facilitated the increased release of soluble and hemolytic

leachates fiom the test rnaterials, resulting in what is most likel y a concentration-related

hemolytic response. This inference is supported by the findings of Syrjanen and associates

(1985) who concluded that water-soluble components from AH 2 8 powder became

increasingl y hem01 yt ic at higher concentrations. When the concentration increased fiom 1

mg/mL to 30 mg/mL, the resulting percent hemolysis increased fiom less than 5% to

approximately 90% hemolysis. The higher level of hemolytic activity compared to findings

of the present investigation may be attnbuted to hemolytic agents within the AH 26@

powder component being more readily released into solution than when allowed to set with

its bis-phenol diglycidyl ether liquid component.

The increased hemolysis observed for AH 26" (with silver) disks compared to that of

the silver-fiee formulation might have been the result of an increased release of silver ions

( ~ g 3 andor titanium dioxide (TiOz). Silver ions have long been known to lyse

erythrocytes (Meneghetti, 192 1 ). Ballinger and associates (1 982) plotted curves

representing the percent hemolysis of three concentrations of erythrocytes (58 x 106, 14 1 x

106, and 334 x 106 erythrocytes per 3.0 rnL volume of isotonic saline + 33 pM sodium

sulphadimidine) by incremental addition of silver cations. When the concentration of Ag'

ions was held constant, the percent hemolysis was inversely related to the number of

erythrocytes. From the slope of each curve it was calculated that, if al1 the ~ g ' ions were

transferred to erythrocytes, hemolysis occurs when the average erythrocyte receives

approximately 1.2 x 10' Ag+ ions. The phosphate buffered isotonic saline used in the

present hemolytic investigation, however, contained chloride salts that may have impeded

the hemolytic action of any silver cations. In the presence of chloride ions (CI-), silver

cations precipitate when the solubility product ([~g+][cl-]; = 1.7 x 10-~* at 25 OC) is

exceeded. The calculated concentration of silver cations attainable in isotonic saline is very

low (about 1.1 nM) and has been cited as a factor impeding investigations on the

pharmacological effects of the Ag+ cation (Ballinger, 1 982).

Titanium dioxide has long been used as a white pigment in cosmetics and foodstuffs

(Thornton, 1927) and is being developed for broad-ranging applications such as a

photocatalytic surface coating capable of destroying foreign organisms such as bacteria,

vimses and molds (Fujishima et al., 1999). This molecule possesses a hemolytic activity

(Zitting & Skyttk 1971) that is related to its concentration (Collan et al., 1986). At a

concentration of 0.5 mg/mL Ti02, Hedenborg ( 1 988) reported hemolytic recordings

reaching 16%. Using sheep blood, Luoto and associates (1997) showed a Ti02

concentration-dependent hemolytic effect ranging fiom less than 5% hemolysis (at 0.5

mghL Ti02) to approximately 15% hemolysis (for concentrations of 5.0 mg/mL TiOz).

Collan and associates (1986) reported a similar relationship between hemolysis and Ti02

concentration.

Two disadvantages regarding the hemolytic testing of dental materials were identified

by Hensten-Pettersen (1 988):

1) there is limited data relating to dental material toxicity available in previous literature

2) artifacts and rnisinterpretations can easily occur when assessing the hemolytic activity of a test material.

The present investigation can attest to these observations by virtue of the number of

methodological modifications required before a satisfactory testing protocol was developed

to meet the specific handling requirements of the test materials and produce meaningfbl,

reproducible results (Appendices 1-9). The range of the standard error of means was

consistent among materials and between repeated tests, suggesting an inherent error within

the testing system. Agglutination of erythrocytes, for instance, resulted in a false, low

hemolytic activity that was rectified by substituting potassium oxalate for a more effective

anticoagulant (E.D.T. A.; Appendices 2 and 3). Haugen and Hensten-Pettersen (1 979)

previously described the erythrocyte agglutination effect and concluded that false negative

hemolytic readings could also result tiom the precipitation of released haemoglobin.

Other potential causes of error in the interpretation of hemolytic test results include

the use of hypotonic solutions in which the blood is incubated, causing extensive hemolysis

even in the absence of any toxic component from the test material. Blood fiom different

animals may produce significant differences in the hemolytic activity of test materials

(Wennberg & Hensten-Pettersen, 198 1). The absorption spectra of dog hemoglobin, for

instance, was shown by Fujisawa and associates (1978) to Vary considerably in the presence

of different extracts of dental restorative resin materials and was also influenced by changes

in pH. The present hemolytic investigation considered another potential artifact; leachable

substances fiom the test materials may have interfered with the spectrophotometric

absorption (at 545 nm A) recorded. An independent evaluation of the leachable, soluble

components compared the optical density of PBS alone to extracts of ~etac-cemm and AH

26@ test samples in PBS (incubated for 90 minutes at 37 OC) to exclude the possibility of

this source of error (Appendix 9).

Other variables, including human and instrument error (e.g. variances in needle gauge

and aspiration rate during blood collection may have affected erythrocyte membrane

fragility, optical density measurements from the spectrophotorneter may not provide true

and consistent readings of free hemoglobin, etc.), must be considered when evaluating the

standard errors resulting fiom t his assay .

Overall, the hemolytic properties of al1 tested materials were comparable to each

other with the exception of AH 26@ (with silver). This material appears to have a

significantly greater hemolytic potential when the concentration of soluble, leachable

products increases in the presence of erythrocytes.

Cytotoxicity of the test materials

Al1 fieshly mixed materials tested in the Millipore Filter assays produced moderate

cytotoxic responses. Over time, t hese materials had a non-cytotoxic effect, consistent wi t h

previous studies indicating a decreased cytotoxicity for endodontic sealers a s the time from

mixing increases (Geurtsen & Leyhausen, 1997).

The prolonged cytotoxic response for AH 26@ is consistent with the findings of

Wennberg (1980), supporting the reproducibility of the Millipore Filter test. At one hour

from mixing, a mean unstained zone diameter of 10.3 (* 1.2) mm ("moderate cytotoxic"

response) was reported while a non-cytotoxic response occurred at six hours from mixing.

Unlike the findings of the present study, however, Wennberg (1980) found AH 26@ to

produce a zone o f "reduced staining intensity" when freshly mixed. He attributed the

increase in cytotoxicity at one hour from mixing to the increase in polymerization products

released during setting.

One explanation for the difference in cytotoxicity of freshly mixed AH 26@ might be

related to differences in the preparation of test materials. Many dental materials (including

~ e t a c - ~ e m " and AH 26@), can be mixed at inconsistent powder-to-liquid ratios since

manufacturers do not provide accurate instructions for mixing ratios. As a result,

characteristics such as hardness and antibacterial properties may be affected. Fuss and

associates (2000) found that AH 26@ (with silver; set for 1 week) was significantly

decreased in hardness when mixed to a lighter (thinner) consistency but relatively

unchanged in its antimicrobial activity against E. faecalis. This resin sealer was most

antimicrobial when freshly mixed, regardless of the powder:liquid ratio used.

The high toxicity of AH 26@ during its setting reaction has been attributed to the

release of formaldehyde (FA; Spingberg et al.. 1993). himal studies suggest FA to

produce damage to the nasal epithelium, possibly resulting in neoplastic development.

There is, however, no epidemiological data indicating a cause-effect relationship between

FA and malignant neoplasms in man. The adverse effects of FA on humans are usually

confined to localized irritation of the eyes, lungs and/or skin (at concentrations > 2% FA)

(ECIETC Technical Report, 1982). Evidence is growing that suggests it is the

concentration rather than accumulated dose that determines the cytotoxic effects of FA on

the nasal mucosa of rats. Ce11 damage and hyperplasia, for instance, do not occur at FA

concentrations below 1 mg/m3 (Environmental Health Criteria, 1989). Heil and associates

(1 996) suggested that the biological evaluation of a material should be less concerned with

the analytical determination of a substance and more focused on establishing the

concentration at which that substance causes h m .

The powder component of AH ~6~ is reported to be biologically tolerated fil vitro by

cultured murine peritoneal macrophages (Syrjanen et al., 1985). This suggests the

cytotoxic effects of AH 26@ materials tested in the present Millipore Filter assay can be

attributed to either the liquid component or to the formation of toxic products of the setting

process ( e g FA). Water-soluble cytotoxins released from set AH 26@ matenal have also

been suspected (e.g. epoxy derivatives of bisphenol-A-diglycidylether) Weil et ai., 1996).

The prolonged "moderate" cytotoxic response produced by both AH 26@

formulations ("with silver" and ccsilver-fiee") tested in the present Millipore Filter study

(see Figure 8) are consistent with the findings of Ferracane and Condon (1990), who

determined that polymenzed resins liberate most unbound substances within 1 day.

Geurtsen and associates (1 998) evaluated the cytotoxicity of AH 26@ (with silver) with

respect to time; after allowing the material to set for 3 days, it was eluted for periods of 24

hours and 5 days. Unbound, irritating substances were released from the test materials

during both elution time periods. Nakamura and associates (1986) found that extracts in

contact with AH 26@ material for 1, 2 and 3 weeks produced severe cytotoxic responses in

vitm. These findings stress the importance in testing different time extracts to elucidate the

long-term cytocornpatibility of endodontic sealers using different testing systems.

Murphy (1988, via personai communication with S. Meryon) suggested that the

surface area of the test material in contact with cells, rather than its volume, is a more

important factor when assessing cytotoxicity. This is an influential consideration when

contemplating the clinical relevance of these resuits. The 7 mm diameter area of exposure

to the HeLa cell monolayer during the MiIlipore Filter testing, for instance, is much larger

than the area of exposure to periradicular tissues through the apical foramen of a root

(approximately 0.50 - 0.68 mm diameter; Kuttler, 1955). In other words, compansons can

be made between materials but their clinical characteristics cannot predictably be

interpreted from the test results due to the large differences in test material surface area

exposed to the ce11 population (Murphy, 1988).

Results of the present investigation suggest the GIC materials to be most cytotoxic

when fieshly mixed, particularly ~ e t a c - ~ n d o . At one hour from rnixing (after the initial

setting reaction) the GIC materials examined (~etac@-~ndo, ~ e t a c - ~ e m @ and ZUT 0.2%)

were "non-cytotoxic". This pattern is consistent with most of the previousl y pu blished

literature, including those of Kawahara and associates (1979) who evaluated two GICs in

ce11 culture. A marked cytotoxic effect at the fieshly mixed stage was attributed to several

possible factors, including the silica, aluminum, calcium, phosphate and fluonde leached

from the glass particles. The polyacid component (e-g. acrylic, maleic, tartaric and benzoic

acids) can also create a decreased pH in the immediate environment, fbrther contnbuting to

a cytotoxic response (Oliva et al., 1996). Beltes and associates (1 997) determined ~etac"-

Endo to possess mild cytotoxicity when allowed to set for six hours, then left in contact

with a fibroblast ce11 line in vitro for periods of 1, 2, and 3 days. Although the specific

cytotoxicity responses for GICs might vary fiom one study to another, this is probably an

indication of the differences in sensitivity of the various testing systems. The overall

cytotoxic trends for GICs are consistent.

Future investigations

Examination of ZUT can continue in the manner recommended in the ANSYADA

biological testing guidelines (1982). Initial screening tests such as the Ames' Test

(mutagenicity) and Styles' Transformation Test (genotoxicity) are acute BI vitro tests that

can provide important information on ZUT matenal prior to proceeding with secondmy in

vivo testing (e-g. the Bone Implantation and Subcutaneous Implantation Tests). This stage

of biological safety evaluation requires the use of animals (e.g. guinea pigs) to test tissue

reactions following material contact over a longer period of time (up to 26 weeks). If these

secondary test results indicate ZUT to be a well-tolerated material then pre-clinical usage

tests (e.g. pulp capping and endodontic usage tests) in dogs are advised before commencing

with clinical testing (ANSI/ADA, 1982; FDI, 1980).

Of a more academic interest, identification and quantification of the soluble,

leachable products contnbuting to the hemolytic and cytotoxic properties of the AH 26@

formulations can be performed. The use of gas chrornatographic and mass spectroscopie

techniques (Sphgberg et al., 1993) andor high-performance liquid chromatography (Koch

et al., 2001) are methods by which the cytotoxic agents can be fùriher elucidated.

Since a purported advantage of ZUT over ~e tac@-~ndo is its enhanced antimicrobial

properties, studies should be conducted to compare their ability to prevent bacterial

colonization of obturated root canals. Furthemore, the quantity of silver ion release over

time, eficacy against various microbial organisms known to contaminate the root canal

system, and the mode of antimicrobial action (e-g. pH changes, fluoride release, silver

cation interactions) are al1 important characteristics of ZUT that need to be investigated.

7) CONCLUSIONS

1. The main findings of this study are that the hemolpic and cytotoxic characteristics of ZUT 0.2% (as determined by the Percent Hemolysis and Millipore Filter tests) are as favorable, or superior, to those of other GECs (Le. ICetacm-~ndo and ~etac-~em@). Results suggest ZUT 0.2% to be less hemolytic and have a shorter duration of cytotoxicity after mixing than the epoxy resin-based AH 26@ sealers ("wit h silver7' and "si lver-fiee9' formulations).

2. AH 26@ (with silver) formulation was significantly (p<O.0001) more hemolytic than the other test materials when the surface area:test mass ratio was increased (by a factor of 2.5). This suggests that the surface area of the test material in contact with the b1ood:saline solution had a significant effect on its hemolytic characteristics, possibly by facilitating the increased release of silver cations and titanium dioxide.

3. Using the Millipore Filter method, the GlCs and epoxy resin-based matenals produced their highest degree of cytotoxicity when fieshly mixed. The GICs were "non-cpotoxic" one hour after mixing while the AH 26@ formulations exhibited a more prolonged cytotoxic response. At six hours &er mixing, a "non-cytotoxic" effect was recorded for the AH 2 6 sealers.

4. Depending on the physical characteristics of the test material ( e g working and setting times), preli minary studies and subseguent modifications to the recommended hemolytic and cytotoxic testing guidelines (ANSVADA, 1982; FDI, 1980) are advised prior to proceeding with the main investigations.

5 . Addition of 0.2% 2eomice ( w t h t . GIC ceramic component) in the formulation of ZUT material did not change the acute, in vitro cytotoxic characteristics of the GIC component (~etac-cema).

APPENDIX 1

RESULTS OF PRELIMINARY HEMOLYSIS TEST (Adhering to FDI and ANSUADA Protocols)

Table 1 summarizes the results of the first preliminary percent hemolysis test using

four endodontic sealers proposed for the main laboratory investigation. A non-parametric

analysis of variance (Kmskall-Wallis test) determined no statistically significant

differences in percent hemolysis of the four sealers (Table 2).

Table 1. First Preliminary Percent Hemolysis Test - Statistical Summary

maximum 1 227.270 1 163.636 1 36.364 1 263.636

sample size mean percent hemolysis standard error of mean median standard deviation variance range minimum

Table 2. Kniskal-Wallis single factor analysis of variance by ranks from preliminary percent hemolysis test (a = 0.05)

Ho: The percent hemolysis of the samples is the same for al1 four endodontic sealer. HA: The percent hemolysis of the samples is not the same for al1 four endodontic sealers.

ZUT 0.2% 2eornica

9 -5 .O5078

46.53029

36.36400 139.59088

19485.61304 454.543 -227.273

chi-sauare 1 8.000 1 8.000 1 7.000 1 8.000 1

KT0308

9 14.14133

32.3941 1

45.45500 97.18234

9444.4063 1 336.363 - 172.727

ZUT 0.2% ~eomic@

asymp. sig. I -433 I .433 .433 I I -429 I

There was no significant difference in percent hemolysis between endodontic sealer types.

~etac@-~ndo

8 -32.95450

17.84905

-27.27300 50.48473

2548.70779 1 54.546

-1 18.182

KT-308

AH 26@ (with silver)

9 90.90878

32.248 13

54.54500 96.7443 8

9359.47541 245 -454 18.182

~etac@'-~ndo AH 26@

The large variances suggested modifications in methodology were required in order

to increase the precision of the assay. Consideration was given to standardizing the surface

area of the materials tested and create unifonn cylinders for test amples.

Materials Used

KT-308 batch #210171

ZUT 0.2% ~eornic* (using KT-308 batch #210 17 1)

AH 26@ (with silver) lot #98 1200084 (powder) lot #98080005 16 (resin)

~ e t a c @ - ~ n d o Aplicap lot #O 1 Y0 10VERW6 1 5

Rabbit blood donor: New Zealand White (NZW) male 9-10 weeks otd 2.2-2.4 kg body mass anticoagulated with 2% w/v potassium oxalate in saline Production room C-0 1, Charles River Canada, St . -Constant, Quebec Canada.

APPENDLX 2

RESULTS OF PRELtMINARY PERCENT HEMOLYSIS TEST

1) STANDARDIZED TEST SAMPLE SURFACE AREA (CYLINDERS)

Table 1. Results of preliminary percent hemolysis test using matenal samples with standardized surface area (cylinder = 340.69 mmz)

40 - I I

Ketac-Endo ZUT 0.2% I

sample size mean percent hemolysis standard error of mean variance minimum maximum

Figure 1. Results of preliminary percent hemolysis test using standardized surface areas of test materials (shown as mean percent hemolysis * S.E.M)

AH 26@ (with silver)

20 -55.8

15.45

4768.9 -1 87.0 34.3

~ e t a c @ - ~ n d o

16 20.09

6.70

718.87 -24.6 76.33

~ e t a c - c e m e

20

ZUT 0.2%

20 -3.69 1

2.89

166.88 -29.02 12.03

12.09

4.35

378.3 1 -28.54 40.35

Figure 2. Evidence o f blood agglutination using potassium oxalate as an anticoaguiant during preliminary Percent Hemolysis Test.

Materials Used

AH 26@ (with silver): powder lot #98 l2OOOMO resin lot #98080005 16

~ e t a c * - ~ n d o Aplicap: lot FW0050056

~etac-cerna: powder lot 44 1 liquid lot 059

ZUT 0.2%: powder lot 44 1 (~etac-cerna) liquid lot 059 (~etac-cerna)

Rabbit Blood Donor: NZW male 3 years, 7 months old 3.5 kg body weight anticoaguhted with 2% w/v potassium oxalate in saline Charles River Canada, St .-Constant, Quebec, Canada

RESULTS OF PRELIMINARY PERCENT EEMOLYSIS TEST

1) STANDARDUED TEST SAMPLE SURFACE M A 2) EDTA ANTICOAGULANT

Table 1, Results o f preliminary percent hemolysis test using cylinders of test material (surface area = 340.69 mm2) and EDTA anticoagulant

AH 2 k (with silver)

sample size mean percent

1 error of mean 1

~ e t a c K ~ n d o

I hemolysis variance standard

Figure 1. Results of preliminary percent hemolysis test using cylinders o f test matenal and EDTA anticoagulant

~etac-cemm

6 4.0

23.88 1.83

ZUT 0.2%

6 -2.55

5 1.86

6 -8.27

2.15 0.60

21.8 1.91

20.17 1.83

Materials Used

AH 26" (with silver): powder lot #9809000230 and lot #9812000340 liquid lot # 98080005 16

~ e t a c @ - ~ n d o : lots FW0050056, FW00495 12, FW00520 18

~ e t a c - ~ e m ? powder lot 44 1 liquid lot 059

ZUT: powder lot 441 (Ketac-~em@) liquid lot 059 (Ketac-~em@)

Rabbit Blood Donor: NZW fernale 22 montfis old 4.0 kg body mass Collected using Vacutainer (Lavender) Becton-Dickinson 7 mL tube volume containing 0.08 1 rnL 15% EDTA (= 12.1 5 mg) using a 20 gauge needle (1 .5" length) Charles River Canada, St .-Constant, Quebec

APPENDIX 4

RESULTS OF PRELIMINARY PERCENT HEMOLYSIS TEST USING MODIFIED PROTOCOL

1) STANDARDUED TEST SAMPLE SURFACE AREA 2) EDTA ANTICOAGULANT

3) BLOOD-SALINE SOLUTION V0LUME:TEST MATERIAL MASS RATIO STANDARDIZED

Table 1. Statistical summary of modified percent hemolysis test results

AH 28 (with silvei) Ketac-Endo Ketic4etn ZUT 0.2%

sample size mean percent hemolysis standard deviation variance standard error of mean (S.E.M.1

Figure 1. Modified percent hemolysis test results using cylinders of matenal, EDTA anticoagulant, and standardized blood volume;test material mass (shown as mean * S.E.M.)

AH 26 6

7.88

3 -4 1

1 1.60 1.39

6 7.97

7.05

49.63 2.88

a 6

2.35

3.34

11.14 1.36

6 5.93

6.33

40.04 2.58

Statistical Analyses:

General Linear Models Procedure Comparing Cylinders of Test Material Class Level Information

Class Levels Values

MATERIAL 4 AH26 KCEM -0 ZUT

Number of observations in data set = 24

Dependent Variable: HEMO

Source DF

Mode1 3

Error 20

Corrected Total 23

R-Square

0.181216

Source DF

MATERIAL 3

Source DF

MATERIAL 3

Sum of Squares Mean Square F Value Pr > F

124.39615713 41.46538571 1.48 0.25 14

562.05503550 28.10275 177

686.45 1 19263

C.V. Root MSE HEM0 Mean

87.88648 5.30 120286 6.03 187500

Type 1 SS Mean Square F Value Pr > F

124.39615713 41 -46538571 1.48 0.25 14

Type III SS Mean Square F Value Pr > F

124.39615713 41.46538571 1.48 0.25 14

The SAS System 10:31 Monday,May7, 2001 3

T tests (LSD) for variable: HEMO

NOTE: This test controls the type 1 comparisonwise error rate not the experimentwise error rate.

Alpha= 0.05 d e 20 MSE= 28.10275 Critical Value of T= 2.09

Least Significant Difference= 6.3844

Means wit h the same letter are not significantly different.

T Grouping Mean N MATERIAL

A 7.968 6 ZUT A A 7.878 6AH26 A A 5.932 6KCEM A A 2.349 6 KJ2NDO

Duncan's Multiple Range Test for variable: HEMO

NOTE: This test controls the type 1 comparisonwise error rate, not the experimentwise error rate

Alpha= 0.05 d e 20 MSE= 28.10275

NumberofMeans 2 3 4 Critical Range 6.384 6.701 6.903

Means with the same letter are not significantly different.

Duncan Grouping Mean N MATERIAL

A 7.968 6 ZUT A A 7.878 6 AH26 A A 5.932 6 KCEM A A 2.349 6 KENDO

Bonferroni (Dunn) T tests for variable: HEM0 NOTE: This test controls the type 1 experimentwise error rate, but generally

has a higher type Il error rate than REGWQ. Alpha= 0.05 d e 20 MSE= 28.10275

Critical Value of T= 2.93 Minimum Significant Difference= 8.9589

Means with the same letter are not significant ly different. Bon Grouping Mean N MATERIAL

A 7.968 6 ZUT A A 7.878 6 AH26 A A 5.932 6 KCEM A A 2.349 6 KENDO

Materials Used

~etac-cemm: powder lot #44 1 liquid lot #O59

~e tac@-~ndo : lot FWOO52O 18

AH 26@ (with silver): powder lot #98 12000340 liquid lot #98080005 16

ZUT 0.2%: powder lot 441 ( ~ e t a c - ~ e m ? liquid lot 059 (~etac-~em@)

Rabbit Blood Donor: NZ W strain, female, 17 months old 4.2 kg body mass anticoagulated: 2 mg EDTA K3/1 mL total blood solution Charles River Canada, St. -Constant, Quebec

One cylinder of material = 340.69 mm2 surface area Incubated with 2 mL of 2% diluted blood-saline solutiodgram test matenal

e.g. test material mass = 0.908 grams volume saline = 1.82 rnL volume diluted blood solution = 0.036 rnL

RESULTS OF REPEATED PERCENT HEMOLYSIS TESTS USING MODIFED PROTOCOL

(CYLINDERS OF MAïXRIAL TESTED)

Table 1. Statistical summary of percent hemolysis test using modified protocol for cylinders of test matenals (first of two experiments)

mean percent 1 10.43 1 8.71 1 5.61 1 9.25

sample size

AH 26 (with silver) AH 26 (silver-free) ZUT 0.2%

hemolysis variance standard error of mean (S.E.M.1

Figure 1. Percent hemolyses of test material cylinders using modified protocol ( show as mean S.E.M.) (first of two experiments)

AH 26 "(with silver)

6

~etac-cerna

6

AH 26 @(no silver)

6

16.97 1.68

ZUT 0.2%

6

19.54 1.80

12.32 1.43

22.56 1.94

Table 2. Statistical summary of percent hemolysis test using modified protocol for cylinders of material (second of two experiments)

----

~ample size mean percent hemolvsis variance standard error of mean (S.E.M.)

silver silver-free

Figure 2. Percent hemolysis of test material cylinders using modified protocol ( s h o w as mean * S. E.M.) (second of two experiments)

Matenal Used

A H 26@ (with silver): powder lot #O04000491 liquid #9911001480

AH 26@ (silver-fiee): powder lot #O00200 1678 liquid #O003000973

~e tac -cema: powder lot 457 Iiquid lot 062

ZUT 0.2%: powder 453 (Ketac-~em@) liquid lot O6 1 ( ~ e t a c - ~ e m ?

Rabbit Blood Donor: NZW female 4.2 kg body mass 23 months old anticoagulated with EDTA K3 (2 mg/mL total volume) Charles River Canada, St. -Constantine, Quebec

APPENDIX 6

RESULTS OF PERCENT HEMOLYSIS TESTS USING MODlFIED TESTTNG PROTOCOL FOR DISKS OF MATERIAL

Table 1. Percent hemolysis - test results using disks of materiai (6 diskdtest sample =

857.22 mmz surface area)

A H 26 (wîh silver) Ketac-Endo Ketac-Cern ZUT 0.2%

sample size

mean percent hemolvsis variance standard error

Figure 1. Results of first percent hemolysis test using disks of material (6 diskdtest sample) (shown as mean * S.E.M.; *p c 0.05)

AH 26@ (with silver)

6

ZUT 0.2%

6

~ e t a c y ~ n d o

6

36.6

299.9 7.07

~ e t a c - c e m W

6

13.0

39.0 2.55

2.96

8.76 2.15

5.95

85.4 3.77

Table 2. Second percent hemolysis test results using disks of material (6 diskdtest sample)

(with silver

percent 1 I hemolvsis 1 1

AH 26@ (silver-free)

1 error I I

-

variance standard

AH 26 (mm slver) AH 26 (olver-free) Ketac-Endo Ketac-Cern ZUT 0.2%

~ e t a c ~ - Endo

- .. -

353.4 7.67

Figure 2. Results of second percent hemolysis test using disks of matenal (6 diskdtest sample) (shown as mean * S.E.M.; *p < 0.05)

- .- - -

134.6 4.72

Ketac- cema

ZUT 0.2%

Materials Used

AH 26' (with silver): powder lot #O0400039 1 liquid lot #99 1 100 1480

AH 269 (silver-fiee): powder lot #O00200 1678 liquid lot #O003000973

~ e t a c - ~ e m ? powder lot 453 liquid lot 062

ZU-T 0.2%: powder lot 453 liquid lot 061

Rabbit Blood Donor: NZW female 4.2 kg body mass 23 months otd anticoagulated with 2 mg EDTA K 3 h L total volume Charles River Canada, St .-Constantine, Quebec

PERCENT HEMOLYSIS TEST STATISTICAL ANALYSES (FOR REPEATED EXPERIMENTS OF BOTE CYLINDERS AND DISKS OF TEST

MATERIALS)

MATERIAL

General Linear Models Procedure Class Level Information

Class Levels Values

9 AHNSCy AHNSDisk AHWSCy AHWSDisk KCEMCy KCEMDisk KendoDis ZUTCy Z U T D i s k

Number of observations in data set = 54

NOTE: Due to missing values, only 48 observations can be used in this analysis.

General Linear Models Procedure Dependent Variable: T E S T l

Source DF Sum of Squares Mean Square F Value Pr > F

Corrected Total

R-Square C.V. Root MSE TEST1 Mean

Source DF Type 1 SS Mean Square F Value Pr > F

MATERIAL 7 0.00067225 3.34 0. 0068

Source DF Mean Square F Value Pr > F

Type III SS

MATERIAL 7 0. 00067225 3.34 O . 0068

General Linear Models Procedure

Bonferroni (Dunn) T tests for variable: TEST1

NOTE: This test c o n t r o l s the type 1 experimentwise error rate but generally

has a higher type II e r r o r rate than Tukey's for a l 1 pairwise comparisons .

Alpha= 0.05 Confidence= 0.95 df= 40 MSE= 0.000201 Critical Value of T= 3.34730

Minimum Significant Difference= 0.0274

Cornparisons significant at the 0.05 level are indicated by v * * * l

Simultaneous

Difference Upper

Between

Limit

0. 033507

O . 033674

O . 036991

0. 037874

O. 047651

O. 052107

O. 058224

MATERIAL Confidence

Compar ison

AHWSDisk - AHWSCy

AHWSDisk - AHNSCy

AHWSDisk - ZUTCy

AHWSDisk - KendoDis AHWSDisk - KCEMCy

AHWSDisk - ZUTDisk

AHWSDisk - KCEMDisk + * *

Simultaneous

Lower

Confidence

Limit Means

AHWSCy - M W S D i s k

AHWSCy - AHNSCy

AHWSCy - ZUTCy

AHWSCy - KendoDis AHWSCy - KCEMCy

AHWSCy - Z U T D i s k

AHWSCy - KCEMDisk

AHNSCy - F H W S D i s k

AENSCy - AHWSCy

AHNSCy - ZUTCy

AHNSCy - KendoDis

AHNSCy - KCEMCy

AHNSCy - ZUTDisk

AHNSCy - KCEMDisk

Z U T C y - AHWSDisk

ZUTCy - AHWSCy

ZUTC y - AHNSCy

ZUTCy - KendoDis

ZUTCy - KCEMCy

ZUTC y - ZUTDisk

ZUTCy - KCEMDisk

General Linear Models Procedure

S imul taneous Simultaneous

Lower Difference UPP-

MATERIAL Confidence Between Confidence

Compar ison Limit Means Limit

KendoDis - AHFISDisk -0.037874 -0.010453 0.016967

KendoDis - AHWSCy -0.031787 -0.004367 0. 023054

KendoDis - AHNSCy -0 .031621 -0.004200 O. 0 2 3 2 2 1

KendoDis - ZUTCy -0.028304 -0.000883 0. 026537

KendoDis - KCEMCy -0.017644 0. 009777 O, 037197

KendoDis - ZUTDisk -0.013187 O , O14233 0.041654

KendoDis - KCEMDisk -0 .007071 0. 020350 0. 0 4 7 7 7 1

KCEMCy - AHWSDisk -0 .047651 -0,020230 0 .007191

KCEMCy - AHWSCy -0,041564 -0,014143 0, 013277

KCEMCy - AHNSCy -0.041397 -0,013977 0,013444

KCEMCy - ZUTCy -0 .038081 -0.010660 O . 016761

KCEMCy - KendoDis -0.037197 -0,009777 O . 017644

KCEMCy - ZUTDisk -0.022964 O, 004457 0. 031877

KCEMCy - KCEMDisk -0.016847 O . 010573 0. 037994

ZUTDisk - AHWSDisk -0.052107 -0,024687 0. O02734

ZUTDisk - AHWSCy -0 .046021 -0,018600 O . O08821

ZUTDisk - AHNSCy

ZUTDisk - ZUTCy

ZUTDisk - KendoDis

ZUTDisk - KCEMCy

ZUTDisk - KCEMDisk

KCEMDisk - AHWSDisk

KCEMDisk - AHWSCy KCEMDisk - AHNSCy

KCEMDisk - ZUTCy

KCEMDisk - KendoDis

KCEMDisk - KCEMCy

KCEMDisk - ZUTDisk

General Linear Models Procedure

Duncan's Multiple Range Test for variable: TEST1

NOTE: This test controls the type 1 comparisonwise rate, not the

experimentwise error rate

error

Number of Means 2 3 4 5 6 8

Critical Range .O1656 .O1741 .O1797 -01837 ,01867 .O1911

Means with the same letter are not significantly different.

Duncan Grouping N MATERIAL

6 ZUTCy

6 KendoDis

6 KCEMCy

General Linear Models Procedure

Bonferroni (Dunn) T tests for variable: TEST1

the type 1 experimentwise error but generally II error rate than REGWQ.

NOTE: This test controls rate,

has a higher type

Alpha= 0.05 df= 40 MSE= 0.000201 Critical Value of T= 3.35

Minimum Significant Difierence= 0.0274

are not significantly different.

Grouping Mean N

Means with the same lettex

Bon MATERIAL

ZUTCy

KendoDis

KCEMC y

ZUTDis k

General Linear Models Procedure Class Level Information

Class Levels Values

9 AHNSCy AHNSDisk AHWSCy AHWSDisk KCEMCy KCEMDis k KendoDis ZUTCy ZUTDis k

Number of observations in data set = 5 4

NOTE: Due to rnissing values, only 47 observations can be used in this analysis.

General Linear Models Procedure

Dependent Variable: TEST2

Source DE S u n of Squares Mean Square F Value Pr > F

Error 0 . 0 0 0 1 8 5 0 3

Corrected Total

Root MSE

Source DE Mean Square F Value Pr > E

MATERIAL 7 0 . 0 0 0 3 2 8 5 1 1 . 7 8 0. 1 2 0 1

C.V.

Type I SS

0.00229956

Source DF Type III SS Mean Square F Value Pr > F

MATERIAL 7 0 .00032851 1.78 O . 1 2 0 1

General Linear Models Procedure

Bonferroni (Dunn) T tests for variable: TEST2

NOTE: This test controls the type 1 experimentwise error rate but generally

has a higher type II error rate than Tukey's for al1 pairwise cornparisons .

Alpha= 0.05 Confidence= 0.95 df= 39 MSE= 0.000185 Critical Value of T= 3.35341

Cornparisons significant at the 0.05 level are indicated by l * * * 1

S imul taneous Simultaneous

Lower Difference UPPer

MATERIAL Confidence Between Confidence

Comparison Limit Means Limi t

AHWSDisk - ZUTCy -0.015932 O . 010403 O . 036739

AHWSDisk - KendoDis -0.013955 O . O12380 O . 038716

AHWSDisk - ZUTDisk -0.014645 O . 012976 O . 040597

AHWSDisk - KCEMCy -0.012321 O . O14015 O . 040351

AHWSDisk - KCEMDisk -0.011818 O . O14518 O . 040854

AHWSDisk - MNSCy -0.003359 0. 022977 O . 049312

AHWSDlsk - AHWSCy

ZUTCy - AHWSDisk

ZUTCy - KendoDis

ZUTC y - ZUTDisk

ZUTCy - KCEMCy

ZUTCy - KCEMDisk ZUTCy - AHNSCy ZUTCy - AHWSCy

KendoDis - AHWSDisk

KendoDis - ZUTCy KendoDis - ZUTDisk

KendoDis - KCEMCy

KendoDis - KCEMDisk

KendoDis - AHNSCy

KendoDis - AHWSCy

ZUTDisk - AHWSDisk

ZUTDisk - ZUTCy

ZUTDisk - KendoDis ZUTDisk - KCEMCy

ZUTDisk - KCEMDisk

ZUTDisk - AHNSCy

ZUTDisk - AHWSCy -0.017104 O . O10517

KCEMCy - AHWSDisk -0 .040351 -0.014015

General Linear Models Procedure

Simultaneous Simultaneous

Lower Difference UPP-

MATERIAL Confidence Between Confidence

Comparison Limit Means Limit

KCEMCy - ZUTCy -0.029947 -0.003612 O . 022724

KCEMCy - KendoDis -0.027970 -0.001635 O . 024701

KCEMCy - ZUTDis k -0.028660 -0.001039 O . O26582

KCEMCy - KCEMDisk -0 .025833 O . O00503 0.026839

KCEMCy - AHNSCy -0.017374 O . 008962 O . 035297

KCEMCy - AHWSCy -0 .016857 0 .009478 0.035814

KCEMDisk - AHWSDisk -0.040854 -0.014518 0.011818

KCEMDisk - ZUTCy -0.030450 -0.004115 o . 022221

KCEMDisk - KendoDis -0.028473 -0.002138 O . 024198

KCEMDisk - ZUTDisk -0.029163 -0.001542 O . 026079

KCEMDisk - KCEMCy -0 .026839 -0.000503 O . 025833

KCEMDisk - AHNSCy -0.017877 O . 008459 O . 034794

KCEMDisk - AHWSCy -0.017360 0. 008975 0. O35311

AHNSCy - AHWSDisk AHNSCy - ZUTCy

AHNSCy - KendoDis

AHNSCy - ZUTDisk

AHNSCy - KCEMCy

AHNSCy - KCEMDisk

AHNSCy - AHWSCy

AHWSCy - AHWSDisk

AHWSCy - ZUTCy

AHWSCy - KendoDis AHWSCy - ZUTDisk

AHWSCy - KCEMCy

AHWSCy - KCEMDisk

AKWSCy - AmSCy

General Linear Models Procedure

Duncan's Multiple Range Test for variable: TEST2

NOTE: This test controls the type 1 comparisonwise error rate, not the

experimentwise error rate

Alpha= 0.05 WARNING: Ce1

Warmonic Mean

df= 39 1 s i z e s of ce11

MSE= 0.000185 are not equal. sizes= 5.853659

Number of Means 2 3 4 5 6 7 8

Critical Range .O1608 -01691 .O1745 .O1784 .O1814 .O1837 . O1856

Means with the same letter are not significantly d i f f e r e n t .

Duncan Grouping N MATERIAL

6 ZUTCy

6 KendoDis

5 ZUTDisk

6 KCEMCy

6 KCEMDisk

6 AHNSCy

6 AHWSCy

General L inea r Models Procedure

Bonfe r ron i (Dunn) T tests for variable: TEST2

NOTE: This test c o n t r o l s t h e t y p e 1 exper imentwise e r r o r r a te , b u t g e n e r a l l y

has a h i g h e r type I I e r r o r rate t h a n REGWQ.

Alpha= 0.05 d f = 39 MSE= 0,000185 C r i t i c a l Value of T= 3.35

M i n i m u m S i g n i f i c a n t D i f f e r ence= 0.0267 WARNING: Ce11 s i z e s a r e n o t e q u a l .

Harrnonic Mean of ce11 s i z e s = 5,853659

Means w i t h t h e same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t ,

Bon Grouping Mean N MATERIAL

ZUTCy

ZUTDis k

KCEMC y

KCEMD i s k

General Linear Models Procedure Class Level Information

Class Levels Values

MATERIAL 9 AHNSCy AHNSDis k AHWSCy AHWSDis k KCEMCy KCEMDisk KendoDis ZUTCy ZUTDisk

N W e r of observations in data set = 54

NOTE: Due to missing values, only 6 observations can be used in this analysis.

General Linear Models P r o c e d u r e

Source DF

E r r o r O . 00013628

C o r r e c t e d Total

R o o t MSE

Source

Dependent Variable : TEST3

Sum of Squares Mean Square F Value Pr > F

C.V.

DF Type 1 SS Mean Square F Value Pr > F

MATERIAL O . .

Source DF Mean Square F Value Pr > F

Type III SS

0

General Linear Models Procedure

General Linear Models Procedure Class Level Information

Class Levels Values

MATERIAL 9 AHNSCy AHNSDisk AHWSCy AHWSDisk KCEMCy KCEMDisk KendoDis ZUTCy ZUTDisk

Number of observations in data set = 54

NOTE: Due to missing values, only 48 observations can be used in this analysis.

MAIN PERCENT HEMOLYSIS INVESTIGATION STATISTICAL TEST RESULTS

(For both cylinder and disk forms of test materials, n = 6; repeated twice, except AH 26" (silver-free) disks, testd once)

General L inear Models Procedure

Dependent Variable: TEST3

Source D F Sum of Squares Mean Square F Value P r > F

Mode 1

E r r o r

Cor rec ted Total

R-Square

Source

C.V.

DE Mean Square F Value

MATERIAL O

Source D F Mean Square F Va lue

O . 0 0 0 6 8 1 4 2

Root MSE

Type 1 SS Pr > F

Type III SS Pr > F

General L inear Models Procedure

General Linear Models Procedure Class Level Information

Class Levels Values

MATERIAL 9 AHNSCy AHNSDisk AHWSCy AHWSDis k KCEMCy KCEMDisk KendoDis ZUTCy ZUTDisk

Number of observations in data set = 54

NOTE: Due t o missing values, only 48 observations can be used in this analysis.

General Linear Models Procedure

Dependent Variable: PERHEMOl

Source DE Sum of Squares Mean Square F Value Pr > F

Mode 1 7 4593 .77602769 656 .25371824 8 .75 0 . 0 0 0 1

E r r o r

C o r r e c t e d T o t a l 47 7 5 9 2 . 2 6 0 9 6 4 5 1

C.V. PERHEMOl Mean

Root MSE

Source DE Type 1 SS Mean Square F Value Pr > F

MATERIAL 656.25371824

Source Mean Square F Value

Type III SS Pr > F

General Linear Models Procedure

Bonferroni (Dunn) T tests for variable: PERHEMO1

NOTE: This test controls the type 1 experimentwise error rate but generally

has a higher type II error rate than Tukey's for al1 pairwise comparisons.

Alpha= 0.05 Confidence= 0.95 di= 40 MSE= 74.96212 Critical Value o f T= 3.34730

Minimum Significant ilifference= 16.732

Cornparisons significant at the 0.05 level are indicated by v * * * i

Simultaneous Lower Di f f erence

MATERIAL Confidence Comparison Limit

AHWSDisk - KendoDis 6,614 * * *

AHWSDisk - AHWSCy 9.177 * * *

AHWSDisk - AHNSCy 9.234 * * *

MWSDisk - ZUTCy 10,358 * * *

AHWSDisk - ZUTDisk 13,665 * * *

AHWSDisk - KCEMCy 13.999 * * *

Sirnultaneous UPPer

Between Confidence Means Limit

AHWSDisk - KCEMDisk 16.694 33.427 50.159 * * *

KendoDis - AHWSDisk -40.079 -23.347 -6.614 * * *

KendoDis - AHWSCy -14.170 2.562 19.294 KendoDis - AHNSCy -14.112 2.620 19.352 KendoDis - ZUTCy -12.989 3.743 20,476 KendoDis - ZUTDisk -9.681 7 .051 23,783 KendoDis - KCEMCy -9.347 7.385 24.117 KendoDis - KCEMDisk -6.652 10.080 26,812

AHWSCy - AHWSDisk -42.641 -25.909 -9.177 * **

AHWSCy - KendoDis -19.294 -2.562 14.170 AHWSCy - PANSCy -16.674 0.058 16.790 AHWSCy - ZUTCy -15.551 1 .181 17,913 AHWSCy - ZUTDisk -12.243 4.489 2 1 - 2 2 1 AHWSCy - KCEMCy -11.909 4.823 21.555 AEfWSCy - KCEMDisk -9.215 7.518 24.250

AHNSCy - AHWSDisk -42.699 -25.967 -9.234 * * *

AHNSCy - KendoDis -19.352 -2.620 14.112 AHNSCy - AHWSCy -16.790 -0.058 16.674 AHNSCy - ZUTCy -15.609 1 .123 17 .856 AHNSCy - ZUTDisk -12.301 4.431 21.163 AHNSCy - KCEMCy -11.967 4.765 21.497 AHNSCy - KCEMDisk -9.272 7.460 24.192

The SAS Sys tem 1 0 5 5 F r i d a y , O c t o b e r 27, 2000 213

G e n e r a l L i n e a r Models Procedure

S i m u l t a n e o u s S i m u l t a n e o u s Lower D i f f e r e n c e Upper

MATERIAL C o n f i d e n c e Between C o n f i d e n c e Compar ison L i m i t Means Limit

ZUTCy - AHWSDisk -43.822 -27.090 -10.358 * * *

ZUTCy - KendoDis -20.476 -3.743 12.989 ZUTCy - AHWSCy -17.913 -1.181 15.551 ZUTCy - AHNSCy -17.856 -1.123 15.609 ZUTCy - ZUTDisk -13.425 3.308 20.040

ZUTCy ZUTCy

- KCEMCy - KCEMDisk

ZUTDisk - AHWSDisk

ZUTDis k ZUTDisk ZUTDis k ZUTDis k ZUTDis k ZUTDis k

- KendoDis - AMWSCy - AHNSCy - ZUTCy - KCEMCy - KCEMDisk

KCEMCy

- KendoDis - AHWSCy - AHNSCy - ZUTCy - ZUTDisk - KCEMDisk

KCEMC y KCEMC y KCEMCy KCEMC y KCEMC y KCEMCy

KCEMDis k - AHWSDisk

KCEMD i s k KCEMDis k KCEMDis k KCEMDis k KCEMDis k KCEMDis k

- KendoDis - AHWSCy - AHNSCy - ZUTCy - ZUTDisk - KCEMCv

SPECTROPHOTOlMETRIC MEASUREMENTS OF TEST MATERIAL SOLUBLE PRODUCTS

Table 1. Summary of optical density readings of test materials (6 diskdtest sample) afier 90 minute incubation (hot water bath at 37 OC) - first of two experiments

Statistical Analyses:

Sample size

General Linear Models Procedure Dependent Variable: OD 1

Source DF Sum of Squares Mean Square F Value Pr>F

Mean OD (545 nm) -0.025 Standard error of 0.001 1 0.0014 O. 0044 mean (S.E.M.)

AH 26" (with silver)

6

Mode1 2 0.00018 178 0.00009089 2.02 O. 1670 Error 15 0.00067450 0.00004497 Comected Total 17 0.00085628

R- Square C.V. Root MSE OD1 Mean 0.2 12288 -22.81719 0.00670572 -0.0293 8889

~ e t a c - ~ e m @

6

Source DF Type 1 SS Mean Square F Value Pr>F MATERIAL 2 0.00018178 0.00009089 2.02 O. 1670

PBS Control

6

Source DF Type III SS Mean Square F Value Pr>F MATERIAL 2 0.000 18 178 0.00009089 2.02 O. 1670

Duncan's Multiple Range Test for First OD Experiment NOTE: This test controls for the type 1 comparisonwise error rate, not the experimentwise

error rate Alpha = 0.05 df = 15 MSE = 0.000045

Number of Means 2 3 Critical Range 0.008252 0.008650

Means with the same letter are not significantly different.

Duncan Grouping Mean N MATERIAL A -0.025167 6 CONTROL A A -0.030167 6 AH26 A A -0.032833 6 KCEM

A Bonferroni (Dunn) T test was conducted: Note: this test controls the type 1 experimentwise error rate, but generall y has a higher type II error rate than REGWQ.

Alpha= 0.05 df = 15 MSE = 0.000045 Critical value of T = 2.69 Minimum signiticant difference = 0.0 104

Means with the same letter are not significantly different.

Bon Grouping A A A

Mean N Material -0.025167 6 PBS Control -0.030167 6 AH 26 -0.032833 6 Ketac-Cern

Table 2. Summary of optical density readings of test materials (6 diskdtest sarnple) afler 90 minute incubation (hot water bath at 37 OC) - second of two experiments

Sample size Mean OD (545

General Linear Models Procedure Dependent Variable: 0D2

Standard error of mean (S.E.M.)

Source DF Sum of Squares Mean Square F Value Pr>F Mode1 2 0.0000121 1 0.00000606 0.20 0.82 13 Error 15 0.00045550 0.0000303 7 Corrected Total 17 0.0004676 1

AH 26" (with silver)

6 -0.028

R- Square C.V. Root MSE 0D2 Mean 0.02590 -19.48737 0.0055 1060 -0.02827778

0.0022

Source DF Type 1 SS Mean Square F Value Pr>F MATERIAL 2 0.00001211 0.00000606 0.20 0.8213

~etac-cemW

6 -0.029

Source DF Type III SS Mean Square F Value Pr>F MATERIAL 2 0.0000 12 1 1 0.00000606 0.20 0.8213

PBS Control

6 -0.027

0.00076

Duncan's Multiple Range Test for Variable 0D2 (second OD test) NOTE: This test controls the type I comparisonwise error rate, not the

experimentwise error rate Alpha = 0.05 df = 15 MSE = 0.00003

0.003 1

Number of Means 2 3 Critical Range 0.00678 1 0.007 1909

Means with the same letter are not significantly different. Duncan Grouping Mean N MATERIAL

A -0.027333 6 CONTROL A A -0.028167 6 AH26 A A -0.029333 6 KCEM

A Bonferroni (Dunn) T Test was conducted:

Alpha = 0.05 df = 15 MSE = 0.00003 Critical Value of T = 2.69 Minimum significant difference = 0.0086

Means with the same letter are not significantly different.

Bon Grouping Mean N

PBS Control AH 26 Ketac-Cern

APPENDIX 10

MILLIPORE FILTER TEST OF CYTOTOXICITY

FORMULATIONS, TECHNIQUES AND DETAILED PRODUCT INFORMATION

Figure 1. Appearance of milüpore lïlter with 24-hour HeLa ceU culture after incubation in tetrazolium blue stain.

Figure 2. Appearance of miilipore filter with 72-hour HeLa cell culture d e r incubation in tetrazolium blue stain.

Figure 3. Increasing coduence of HeLa ceii monolayer cultured on millipore iilter disks &er incubation periods of (a) 24 hours, (b) 48 hours, and (c) 72 hours.

a

Histochemical Preparation of Filters for Light Microscopy:

1) Microscopic evaluation of HeLa confluence on millipore filters (adapted from Wennberg, 1988)

The filter (with the adherent ceIl layer) is gently rinsed with PBS solution and

transferred to a solution of either 3% glutaraldehyde or 10% formalin for a period of 1 5

minutes to achieve fixation of cells. The fixed specimens are then stained with

haematoxylin and eosin and the filter is then cut into pieces approximateiy 1 cm2. These

pieces are immersed in xylene to render the filter transparent and then mounted on slides

with the ce11 layer facing the cover slip.

2) Evaluation of HeLa monolayer adherence to millipore filter (cross-sectional view)

Millipore filters with stained ce11 monolayers were loosely folded over once to

maximize surface area observed pnor to embedding in wax and sectioning. The filter

samples were embedded in surgiPath@ Blue Ribbon TissuelInfiltration Medium (Reorder

No. 61330) and sectioned to 7 micron widths using a microtome. The sections were then

rnounted on glass slides and subjected to the following staining procedures (fiom Bancroft

and Stevens, 1990):

Xylene solution submersion for 3 minutes, repeated 3 times. 100% ethanol submersion for 2 minutes, repeated twice. 95% ethanol submersion for 2 minutes, repeated twice. 70% ethanol submersion for 2 minutes, repeated twice. 50% ethanol submersion for 2 minutes, repeated twice. Distilled water submersion for 2 minutes, repeated twice. Stained with Harris hematoxylin (Fischer Brand) for 6 minutes. Rinsed in distilled water. Differentiated in 1% HCI in 70% ethanol (one or two drops). Rinsed in distilled water. Stained in Eosin Y (Sigma Brand) for 1 minute. Rinsed in distilled water. Mount in Kaiser's glycerol gel prior to mounting.

Detailed Product Information:

HeLa (ATCC number CCL-2): for culturing information see ATCC website htt~://phage.atcc.org/cgi-bidsearchen ine/longview.cgi?view=ce.26767&text=Hela

AH 26@ (with silver): powder lot #98 l2OOO34O,99OlOOO28 1,9902000524,000400049 1 liquid lot #98 l2OOO340,990 100028 1,990 10008 19,991 100 1480

AH 2 6 (silver-free): liquid lot #O03000973

ZUT 0.2%:

Wax controls: Dental boxing wax, The Hygenic Corporation Akron, Ohio 443 10 USA

powder lot #O003 00049 1

lot FWOO495 12, FWOOS2O 18, FWOO6O 146

powder lot 438, 457 liquid lot 059,062

Ketac-cerna powder lot 44 1,457 Ketac-Cern' liquid lot 059, 062

ID#00816 032894 303

Lactated Ringer's Solution:

Lot (L)52- 126-NA 273 mOsm/L pH 6.7 mm01 (rnEq)/L : Na 130

K 4 Ca 1.5 Cl 1 09 Lactate 28

100 mL: sodium lactate anhydrous 0.3 I g sodium chloride 0.6 g potassium chloride 0.03 g calcium chloride dihydrate 0.02 g

Abbon Laboratories Ltd. St.-Laurent Quebec H4S 121

Agar Medium Formulation: (100 mL total volume provides enough to make 20 petn dishes; SmWdish)

73 mL a - MEM (Eagles) 1 -5 g BactoAgar 10 mL antibiotics (1 0x concentration) 1 mL HEPES (= 10 mM = 1 N l O O mL medium) 1.12 mL sodium bicarbonate (= 10 rnM) 15 mL fetal calf serum (FCS)

BactomAgar @ifcoa brand; Sparks, MD) Lot 139401 XC 454 g Becton Dickinson Microbiology Systems Becton Dickinson & Co. Sparks MD 2 1 1 52 USA

Formulation for 10x antibiotics solution (200 mL total):

20 m t x 10% FCS 10000 U penicillin (50 U h L ) 10 000 ug gentimycin (50 pg1m.L) 50 pg Fungizone (amphotericin B) (0.25 pg/mL)

Formulation for a-MEM growth medium (500 mL total):

50 mL x 10?/oFCS 50 mL, antibiotics (10x concentration) 5.6 rnL sodium bicarbonate buffer 5 mL HEPES buffer 398.4 rnL a-MEM media

Nitro blue tetrazolium chloride monohydrate: see www. sigma-aldrich.com/SACa (Sigma product number N6639)

Formula : C4oHdJ2N , 0 0 6

Formula weight: 8 17.6 Ap pearance : yellow powder Solubility : clear dark yellow solution at 10 mg/mL in water Suitability : suitable for detection of alkaline phosphatase conjugates in

nucleic acid probe detection systems. 50 mghottle pH set to 7.3 after diluting to 0.2% solution (= 100 mg NBT in 50 rnL distilled water)

Sigma Chemical Co., F.O. Box 14508 St. Louis, MO 63 178 USA Phone: 3 14-77 1-5750

Succinic acid (butanedioic acid) disodium salt hexahydrate Sigma product number (S-9637) lot 36H087iS C4&04Naz-6 Hz0 F.W. 270.1

Diluted in distilled water (to 0.06 M solution) and p H set to 7.0; stored in sterile bottle covered with foi1 at 4 OC.

catalogue number HATF04700, lot number H9AM50723 1 001pack 0.45 pm pore size, white surfactant fiee, HATF 47 mm diameter EEC #6O3 -03 7-0 1 -8 Milli pore Corporation Bedford, MA 01 730

Glass Rings Contact: Mr. Fred Leslie

Chemical Engineering Glassworks Laboratory 200 College Street, Room 227 University of Toronto Phone: 4 16-978-306 1

~eflon" Molds Contact: Mr. Dave Powell

Wahlberg Building Machine Shop 200 College Street University of Toronto Phone: 4 16-798-046

APPENDIX 11

MILLIPORE FILTER TEST OF CYTOTOXICITY STATISTICAL SUMMARIES

General Linear Models Procedure Comparinn Test Materials When Freshlv Mixed

Dependent V a r i a b l e : TRIALMN

Source DF S m of Squares Mean S q u a r e F V a l u e P r > F

Error 0.03219444

C o r r e c t e d Total 59 737.96083333

R - S q u a r e Root MSE TRIALMN Mean

Source DF Mean Square F V a l u e P r > F

C . V .

T y p e 1 SS

MATERIAL 5 736,22233333 147,24446667 4573.60 0 .0001

Source DF Type III SS Mean Square F V a l u e P r > F

MATERIAL 5 736,22233333 147,24446667 4573.60 0 .0001

General Linear Models Procedure

Duncan's Multiple Range Test for variable: TRI-

NOTE: This test controls the type 1 comparisonwise error rate, not the

experimentwise error rate

Number of Means 2 3 4 5 6 Critical Range .1609 -1692 ,1747 ,1787 .1818

Means with the same letter are not significantly different.

N MATERIAL

10 KEFresh

10 ZUTFresh

10 KCEMFres

Duncan Grouping

General Linear Models Procedure Comparing Test Materials 1 Hour From Mixing

Class Level Information

Class Levels Values

MATERIAL 5 AHNSlhr AHWSlhr KCEMlhr KElhr ZUTlhr

Number of observations in by group = 50

The SAS System General Linear Models Procedure

Dependent Variable: TRIALMN

Source DF Mean Square F Value Pr > F

Model 4 169.80230000 7571.07 O. 0001

Error 45 0.02242778

Corrected Total 49

R-Square Root MSE TRIALMN Mean

Source DF Mean Square F Value Pr > F

MATERIAL 4 169.80230000 7571 .O7 O. 0001

Source DE' Mean Square F Value Pr > F

Surn of Squares

C.V.

Type 1 SS

Type III SS

4 679.20920000 7571.07 o. 0001 General Linear Models Procedure

Duncan's Multiple Range Test for variable: TRIALMN

NOTE: This test controls the type 1 comparisonwise error rate, not the

experimentwise error rate

Number of Means 2 3 4 5 Critical Range ,1349 - 1 4 1 9 ,1464 ,1497

Means with the same l e t t e r are not significantly different.

Duncan Grouping Mean

10 AHWSlhr

General Linear Models Procedure Comparing Test Materials 2 Hours From Mixinq

Class Level Information

Class Levels Values

Number of observations in by group = 50

General Linear Models Procedure Dependent Variable: TRIALMN

Source DF Sum of Squares Mean Square F Value Pr > F

Correc ted To ta l 4 9 655.36480000

R-Square Root MSE TRIALMN Mean

C . V .

Source D F Type I SS Mean Square F Value Pr > F

MATERIAL 4 652.28330000 163.07082500 2381 .37 O, 0 0 0 1

Source DF Type III SS Mean Square F Value P r > F

MATERIAL 4 652.28330000 163.07082500 2381.37 O . 0 0 0 1

General Linear Models Procedure

Duncan's Multiple Range Test f o r variable: TRI-

NOTE: This

Means

test controls the type 1 comparisonwise error r a t e , not the

experirnentwise e r r o r rate

Number of Means 2 3 4 Critical Range ,2357 .2479 .2559 .2616

with the same letter are not significantly different.

MATERIAL

AHNSShr

Duncan Grouping Mean

General Linear Models Procedure Comparing Test Materials 3 Hours From Mixing

Class Level Information

Class Levels Values

MATERIAL 5 AHNS3hr AHWS3hr KCEM3hr K E 3 h r ZUT3hr

Number of observations in by group = 50

General Linear Models Procedure

Dependent Variable: TRIALMN

Source DF Surn of Squares Mean Square F Value Pr > F

Error O . 04849444

Corrected Total 4 9 778 .71145000

R-Square C.V. Root MSE TRIALMN Mean

Source DF Mean Square F Value Pr > F

MATERIAL 4 1 9 4 . 1 3 2 3 0 0 0 0 4003 .19 O . 0 0 0 1

Source DE Mean Square F Value Pr > F

MATERIAL 4 1 9 4 .13230000 4003 .19 O, 0 0 0 1

Type 1 SS

7 7 6 . 5 2 9 2 0 0 0 0

Type III SS

776.5292OOOO

General Linear Models Procedure

Duncan's Multiple Range Test for variable: TRIALMN

NOTE: This test controls the type 1 comparisonwise error rate, not the

experimentwise error rate

Number of Means 2 3 4 5 Critical Range -1984 , 2 0 8 6 - 2 1 5 3 - 2 2 0 2

Means with the same letter are not significantly different.

Duncan Grouping Mean N MATERIAL

General Linear Models Procedure Comparing Test Materials 6 Hours From Mixing

Class Level Information

Class Levels Values

MATERIAL 5 AHNS6hr AHWS6hr KCEM6hr KE6hr ZUT6hr

Number of observations in by group = 51

General Linear Models Procedure

Dependent Variable: TRIALMN

Source DF S m of Squares Mean Square F Value Pr > F

Mode 1 4 O O

E r r o r O

Corrected Total 50 O

Root MSE R-Square TRIALMN Mean

Source DI' Mean Square F Value Pr > F

Source DF Mean Square F Value Pr > F

C.V.

Type 1 SS

Type III SS

General Linear Models Procedure

Duncan's Multiple Range Test for variable: TRIALMN

NOTE: This test controls the type 1 comparisonwise error rate, not the

experimentwise error rate

Alpha= 0.05 df= 46 MSE= O WARNING: Ce11 sizes are not equal.

Harmonic Mean of ce11 sizes= 10.18519

Number of Means 2 3 4 5 Critical Range O O O O

Means with the same letter are not significantly different.

Duncan Grouping Mean N MATERIAL

General Linear Models Procedure Comparing Test Materials 24 Hours From Mixing

Class Level Information Class Levels Values

Source

Number of observations in by group = 50

General Linear Models Procedure

Dependent Variable : TRIALMN

E r r o r

DF Sum of Squares Mean Square F Value Pr > F

Correc ted T o t a l

R-Square C.V. TRIALMN Mean

Source DF Mean Square F Value

MATERIAL 4 O 0

Source DF Mean Square F Value

O

Root MSE

Type 1 SS Pr > F

Type III SS Pr > F

MATERIAL

General Linear Models Procedure

Duncan's Mult ip le Range Test f o r v a r i a b l e : TRIALMN

NOTE: T h i s t e s t c o n t r o l s t h e type 1 cornparisonwise e r r o r r a t e , no t t h e

experimentwise e r r o r r a t e

Number of Means 2 3 4 5 C r i t i c a l Range O O O O

Means wi th t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t .

Duncan Grouping N MATERIAL

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