Reiiabiiity of in Vitro Microleakage Tests:A Literature Review

Anne RaskinVWilliam D'Hoore^Samuel GonthierVMichel Degrange'^/Jacques

Purpose: The literature contains conflicting data about in vitro microleakage evaluations and their usefui-ness and reiiabiiity. No standardization has yet been established. Here we consider features of publishedstudies that might affect the results of the in vitro microleakage tests.

Materials and Methods: We reviewed 144 in vitro microleakage studies, published in 14 internationai re-views between 1992 and 1998, which comprised 917 sets or groups of experiments. The published stud-ies were entered in a database and compared using seiected literature criteria: sampie, cavities,restoration procedures, thermocycling and mechanical cycling, evaluation method.

Results: The methods employed vary widely. The most frequent methodologicai choices (%) were (1) speci-men storage after extraction: duration (unspecified, 59.2|, medium (distilied or deionized water, 33,8),temperature ¡unspecified, 52.2), additives (none, 47.0); (2) aging method (79,1): duration before aging(< 24 h, 35,9); medium and temperature of storage before aging (distilled or deionized water, 26.8; 37°0,54.3): (3) medium of cycling (tap water, 50.5), number of cycies ([250-500], 34.6), number of baths (2,84.0), bath temperature (5°C to 55°C, 60.6), immersion dwell time (30 s, 44.3); (4) tracer; type (basicfuchsin, 40.7), time of immersion (after thermocycling and/or mechanicai cycies, 64.1), immersion dura-tion ¡basic fuchsin: 24 h, 59.5); assessment of dye penetration of sections (91,7); direction (perpendicu-lar, 88.5), number ¡1, 47.1).

Conclusion: The great variability in the methods used in these 144 studies prevented meta-analysis andcomparison of the results, thus reducing the value of these methods,

J Aühesive Dent 2001:3:295-308. Submitted for pubiication:13.03.01: accepted for publication:23.08.01.

The interface between restorative material andtooth structure is known to be an area of clinical

concern that can result in marginal discoloration.

a Resident, Department of Operative Dentistry and Endodontics.Sctiooi of Dentistry. Université cathoiique de Louvain, Brusseis.Belgium.

" Professor, School of Public flealth. Université OatfioiiQue de Lou-vain, Brusseis, Beigium.

^ Ciinical Assistant, Department of Biomaterials. Faculté de chirur-gie dentaire. Université de Paris V, France.

" Professor and Head, Department of Biomateriais, Facuité dechirurgie dentaire. Université de Paris V. France.

« Professor and Head, Departmeht of Biomateriais. Faculté d'odon-tologie. Université de /a Méditerranée. Marseille, France.

Reprint Requests: Anne Rsskin. Ecole de Médecine Dentó/re et deStomatologie. Université Cathoiique de Lcuvain. Av. Hippocrate, 15.B-1200 Bruxelles, Belgique. Tel: *32(0)2-764-57-40. Fax: '•32-10)2-764-57-27, e-mail: raskln®patd.ucl.ac.be

secondary caries, and pulpal pathology,^ Theturnover of restorative materials, especially dentinbonding agents and composite resins, is substan-tial, and it is therefore impossible to test all thesematerials olinioally. In vitro tests remain an indis-pensable method of initial screening of dental ma-terials and set a theoretical maximal amount ofieakage that may or may not occur in vivo.2' Themost common method of assessing the sealing effi-ciency of a restorative material is microleakageevaluation. Between 1992 and 1998, more than300 studies (Medline) on microleakage were pub-lished in the literature. Unfortunateiy, these studieshave generally given contradictory results which areall the more difficult to interpret since each studyevaluated a limited number of materials. Thesecontradictions are probably due to differences intechnicai procedures and, as yet, no standardiza-

Vc'295

Raskin étal

Table 1 Inclusion and exclusion selection criteria

Study

Sample

Cavity preparation

Materials

Inclusion entena

Microleskagein vitro tests

Human or bovine teeth

Operative andprosthetic dentistry

Resin corrposite, glass ionomer,amalgam, compomer, ceramic,...

Exclusion criteria

Literature review

Endodontics

Table 2 Alphabetical list of publication journals

Publicaticn journals

American Journal cf DentistryAustralian Dental JournalCaries ResearchDental MaterialsInternational Journal of ProsthodonticsJournal of the American Dental AssociationJournal of DentistryJournal of Dental ResearchJournal of Oral RehabilitationJournal of Prosthetic DentistryOperative DentistryPédiatrie DentistryQuintessence InternationalSchweiz Monatsschrift Zaiinmedizin

MATERIALS AND METHODS

We appiied the five steps described by Cohen^ form eta-ana lysis:

1. Specifying inclusion criteria: Criteria for the firstselection of studies are listed in Table 1.

2. Locating studies: The Mediine database wassearched for potentially relevant studies.

3. Seiection of pubiications: Studies on in vitro mi-croleakage tests published between 1992 and1998 were included. Eourteen journals were cho-sen and are listed in Table 2.

4. Coding study features: Restorative materiais,techniques used, and methodological factors po-tentially affecting in vitro microieakage resultswere recorded in a database using selected iiter-ature criteria (Table 3).

5. Final exclusion: Studies suffering from method-ological problems were excluded from this re-view.

tion has been established, Retief in 1991 and Stan-ley in 1993 pointed out the same problem with thebond strength studies.S'̂ '̂ '̂ In the same way, Hiltonhas recently grouped together and identified someexperimental conditions that could affect the mi-crcleakage evaluations.^^

Consequently, an overview of restorative proce-dures is difficult to establish, thus decreasing thereliability and value of these microleakage tests.

The present literature review was designed to in-ventory restorative materiais and techniques, andto identify methodological factors that might poten-tially affect the results of in vitro microleakagetests.

RESULTS

We recorded 144 studies comprising 917 groups ofexperiments (range: 1 to 33 groups/study). Themost frequent choices for restorative materials,techniques, and methodological factors are pre-sented in Table 3.

Sample Characteristics

The most frequent choices were human teeth(96.0%), molars and third moiars (49.6%), and tensamples per group (44,0%) (Fig 1). The tooth stor-

296 The Journal of Adhesive Dentistry

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Table 3 Mostfrequent choice of the restorative materials, techniques, and metho do i ogica i factors registered

Most frequent ehoice

SampiesSubstrate and tooth morphology

Reason for extractionNumber of teeth in each sampleStorage medium atter ertraetion

Temperature tooth storage after extractionTooth storage duration after ertraetion

CavityCavity form

Dimensions

LocationBevel

Res fora tii/e procedureDirect

Light curing

Cervieai matrix

Aging

If no aging

If aging

HumanMolars and third moiarsunspeeified10Distiiied or deionized waterNo additiveUnspecifiedUnspecified

Ciass V(U, Cor V-shaped)

Ciass V

Cemento-enamei junctionNo

Restorative materialDentin bonding agentsBase or liner

Materiais tested

Total durationFilling technique

Ciass V(n = 573)Ciass II (n = 213)

Yes

Ciinicai serviceMedium storage

Duration after specimenfabrication

Storage medium beforeeyeiing

Duration before agingMedium of eyeiing

U-shapedHeight: 2 mmWidth : 3 mmDepth : 1.5 mm

Resin eompositeYesNone

Resin composite>60sIncrementsincrements(resin composites)NoneYes

NoNoneTem pe rature ^ y C

No

Distilled ordeionized water,Temperature:37''C< 24 hoursTap water

GrouDSNumber

880455790403310431479543

573410/573233/573249 / 573277 / 573600617

803526/803581 / 803668/803

608

526/608195 / 608382 / 608

362 / 526439/573108/213

725

192

165/19268/19277/ 124

61/ 192

725

194/725342/630260/725366/725

%

96.049.686.244.033.847.052.259.2

62.571.640.743.548,365.467.3

87.665.572.483.2

66.3

86.532.162.8

68.876.650.7

79.1

21.0

87.535.462.1

31.8

79.1

26.854.335.950.5

Vpl ^ Nn ä. 2001297

Rashin et al

Table 3 (continued)tors registered

Most frequent choice

Thermai cycling

Mechanicai cycling

Duration after aging

Dye/tracerType

pHMoment of immersion

Microleakage evaluationEvaluation methodEvaluation criteriaSections

Statistics

Statistical testNonparametric

_____ . ^

Most frequent choice of tiie restorative materiais, techniques, and methodoiogicai fac-

Number of cyciesNumber of bathsDwell time ofimmersionBath temperature

Number of oyciesForce

Basic fuchsinConcentrationimmersion durationUnspecifiedAfter aging

2 dimensionsScoresYesNumOerDirectionUnspecified

[250-50012

30 s5°Cand55°C

5000125 NNone

0.5%24 h

1Perpendicular

firouDSNumber

705/725244/705592 / 705

312/705427/70559/72517/ 5918/ 59

638 / 725

373251/373222/373

588

813660837396/837744/837282

604

%

97,2

34,684,0

44,360,68,1

28,830-588-0

40.767,359-5

>95,064,1

88,772,091,347,388,930,8

65,9

Unspccpifcü

16 lo 20

1 1 [K 1 i

1 \"

16.1

2L.SF i g l Numberofteeth in expéri-mentai groups (%),

298 TheJournai of Adhesive Dentistritry

Raskin et

Fig 2 Cavity type (%].

age medium after extraction was distilled or deion-ized water (33.8%) and no additives were used(47.0%). Tooth storage temperature and durationafter extraction were unspecified in 52.2% and59.2% of groups, respectively.

Cavity Type

Olass V cavities (Fig 2) were used (62.5%) (U-shap-ed: 71.6%). Cavities were preferentiaily located atthe cementoenamel junction (65.4%) and no bevef(67.3%) was made at the enamel margins.

Class V dimensions were 2 mm high (40.7%), 3mm wide (43,5%), and 1.5 mm deep (48.3%).

Restorative Procedure

Direct restorative procedures were tested in 87.6%of groups (n = 803), and resin composites (65.5%)and dentin bonding agents (72.4%) were the mosttested mater ia ls . No base or i iner was used(83.2%).

Among light-cured materials (n = 608; 66.3%),total light-curing duration was > 60 s (32.1%) andthe materials were applied in increments (62.8%).Only light-cured resin composites were used andthe inorementai technique (cf. bulk technique) forthis material was applied in 62.8% of groups.

No matrices were used for Ciass V (76.6%) or forClass II (32.4%) cavities.

Aging

79.1% of the groups included an aging technique.

Without aging (n = 189)

12.5% of groups were realized and evaluated afterclinical service. The restorations were either evalu-ated directiy with no storage (35.4%) or after stor-age at 3 7 ^ (62,1%).

IVftfi aging

97.2% of groups were thermally cycled (Fig 3). Stor-age medium before cycling was distilied or deion-ized water (26.8%) and storage temperature was37°C (54.3%). Before aging, the teeth were kept inthe storage medium for less than 24 h (35.9%).Teeth were generally thermaliy or mechanicaliy cy-cled in tap water (50.5%). Among the thermocyciedgroups, more than 250 and fewer than 500 cyclesof thermal stress (Fig 4) were used in 34.6% in twobaths (84.0%) with a dwell time of immersion of 30s (44.3%). The minimai and maximal bath tempera-tures were 5°C and 55"'C (60.6%). Only 8 .1% (11studies) of groups used mechanical cycling (Fig 3).The number of cycles ranged from 100 to 500,000and the forces applied on the restorations variedbetween 72.5 and 340 N.

Vol 3. No 4, 2001 299

Raskin et al

nu

n ̂ 725

i-» a.!

41 1

Fig 3 Type of aging (%).

UnspceiHed [1 0.4

>2U00

IIIHWK. LSOOI

1750 lo lOOOl

1500 10 7501 1 1 3.4

[250 lo 5001

[0 lo 250)

13,1

12.9

8.2

m

34.6

24.3

Fig 4 Number of thermal cycles (%).

Dye/Tracer

In 64.1% of cases, teeth were immersed in dye/tracer solution after thermal and/or mechanical cy-ciing (Fig, 5). The three most frequently used dyes/tracers were basic fuchsin, méthylène blue, and sil-ver nitrate in 40.7%, 22.0%, and 17,0% of the

groups, respectively (Fig 6), Immersion durationwas generally 24 h for fuchsin, 2 hours for silver ni-trate, and 4 hours for méthylène blue. The pH ofthe dye solution was unspecified in more than 95%of the groups.

300 The Journal of Adhesive Dentistry

Fig 5 Time of immersion in dye/tracer (%).

With ind uHa Ihünmil iindlnrincch;inlcil cyclih

L(vlf.

WilhQiit if ing

cycio

.VÏ

1 5

KÍ.+ ri

18.2

64.1

Procein red

Cryslai violet

Suffered procion (.rillijnt red

Knoûjmiiie i

Cyrbon dve 1

Neuiral red 1

Unspecified 1

Lresvlblue 1

lolucncbluc 1

Ërvih rosin 1

1 U y

1 J.U

1 1 1

i

i

1.3

1.3

!J.I

1

J 3.1

None

Silver nilrale

Melhylene blue

Basic fue h sin

H 17-0

Fig 6 Type of dye/tracer (%).

Microleakage Evaluation

The most common method was two-dimensionaievaluation (88.7%), using ordinal and dichotomicdata (72.0%), Sections of restored teeth were madein 91.7% (n = 841) of the groups. Only one sectionwas made in 47.1% of groups and two or three sec-

tions were made in 20.0% and 12.7% of groups, re-spectively (Fig 7). Sectioning was perpendicuiar tothe restoration in 88.5% of the groups.

The statistics (ie, mean, maximum) of dye/tracerpenetration were unspecified in 30.8% of cases.Nonparametric statisticai analysis was appiied in65.9% of cases.

vol 3. No 4, 2001 301

Rasiíin et ai

25 1 1 0" L

30

4 or 5

5

Unspeeified

4

3

2

1

07

1.0

1.0

n = Si7

4.9

Scale: log (>;-^ i)

i l .y

I2.S

20.1

47 3

Fig7 Number of sections (%).

Only three of these 144 studies (21 experimentalgroups; 2.3%) specified a\\ restorative materiais,techniques, and methodological factors. The per-centage of unspecified groups ranged from 0 to59.2%. When we tried to pool groups with morethan two or three similar methodological factors (ie,cavity shape, restorative materials, and type oftracer), we always obtained fewer than 15 groups,and meta-analysis was therefore impossible.i"

DISCUSSION

It is difficult to affirm that the 144 studies reviewedhere constitute all studies published between 1992and 1998 in the 14 journais considered. Neverthe-less, the search procedure (Mediine database)likeiy yielded most of the published studies.

These 144 studies comprised 917 groups of ex-periments. The results were expressed in percent-ages of groups and not in percentages of studies,firstly to avoid underestimating the statisticai

weight of studies with the largest number of groups(range: 1 to 33), and secondly to take into accountthe different study objectives, methodologies, andmaterials evaluated.

Criteria for the first selection of studies (Table 1)were determined to increase the internal validi-ty and just to compare studies of microleakage.Restorative materiais, techniques used, and me-thodoiogicai factors were recorded to determine bymeta-analysis which variables of these proceduresoould influence the results, and to compare the ma-teriais used in these studies. However, the studiesshowed a pronounced degree of non-homogeneity.Furthermore, restorative materials, techniquesused, and methodological factors were not alwaysspecified (0 to 59.2% of unspecified groups). Con-sequently, small groups were obtained when tryingto gather studies with two or three similar method-oiogicai factors, and the conditions needed for thestatistical tests of meta-analysis were not met.̂ -̂'

This being so, it was impossible to know whichsteps of the procedure should be taken into ao-

302 The Journai of Adinesive Dentistry

Raskin et ai

count in the variations of the results recorded in thedifferent studies and when comparing the materialstested. The only thing we know is the influence ofsome methodological factors tested in several stud-ies; unfortunately, these were tested separately,and the results were also sometimes contradictory,as discussed in the following.

Sample Size

Sample size was less than or equal to 10 in 60% ofcases, and less than 20 in more than 90%. Thissmail sample size limits the choice of statisticaltests that might be used. Indeed, only nonparamet-ric tests could be used, and these are less powerfulthan parametric tests.i^

Cavity Shape

Cavity design may influence the microleakage re-sults. The spherical Class II restorations had nar-rower marginal gaps than the did rectanguiarrestorations.^ and the U-shaped Glass V cavity wassuperior to the V-shaped design in reducing mi-croleakage,i"*

Light Curing

Light-curing conditions (variable iight intensity, ex-posure times, increments) significantly influenceddirect composite restorations5.2i,29,33 gnd were sei-dom described and controlled.

Aging

To simulate clinicai conditions, restorations weregenerally subjected to thermai stress and/or oc-clusal loads. The most frequently used method wasthermal cycling, which simulates temperature varia-tions in vivo. The influence of aging was tested byseveral authors and the resuits were conflicting.Some authors demonstrated that neither thermocy-cling nor occlusal stress increased the microieak-age of the restorations,8.22.28,34,36,39 othersreported that the need for thermocyciing is depen-dent either on how thermally conductive therestorative is in reiation to its mass,̂ ^ or on the ma-teriais.'^•^^'^^ In contrast, some authors showed

that thermocycling increases ieakage at the dentin-cement interface "̂̂ and that microleal<age was sig-nifjcantiy greater when the restorations weresubjected to both temperature cycling and ccciusalloading, compared to restorations subjected to ei-ther temperature cycling or load cyciing,^^ Never-theless, one study demonstrated that tensiie andcompressive load cycling did not add significantiy tothe microieai<age effects of thermocycling,!" Galeand Darveil conciuded in a literature review^^ thatthermal stressing of restoration interfaces is oniy ofvalue when the initial bond is already known to bereliable.

Tracer

The great majority of microieakage studies examinepenetration of a tracer at the tooth/restoration in-terface. This penetration may be affected by thetracer's particle size or pH,̂ ^ However, Youngson etal noted no difference between four tracers, de-spite a wide range of pH,3s

Penetration may aiso depend on the tracer's con-centration and its diffusion coefficient, the thick-ness of the dentin, and the surface area of thedentin available for diffusion.^o Use of tracer maylead to overestimation of microieakage because ofpermeability of dentin tu bules,1= One study showedthat the radioisotope test generally indicated agreater degree of leakage than did the uitravioletdye test,2 whereas another study showed a closeagreement between the two methods,3i

Evaluation Method

Microieakage is not uniform along the circumfer-ence of a restorative margin,̂ 3,32 and may be moreextreme at end surfaces,!^ Three-dimensionai evai-uation revealed more severe leakage than a con-ventional, single longitudinal, midiine sectioningtechnique^^ or a few sections,ii However, three-di-mensional evaluations are not easy, and the tech-nique is user sensitive (ie, clearing protocol) andtime consuming.̂ 5

Evaluation using sections

Single sections seem to be insufficient for reliabledetection of the deepest tracer at a tooth-restora-

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Raskin et ai

tion interface of Class V cervical margin restora-tions. The use of three sections may avoid under-estimation of in vitro microleakage.̂ 2

Despite the contradictions, it seems evident thatthe sealing ability of restorative materials is highlydependent upon the procedure employed, and it isimpossibie to evaluate the role of each study fea-ture. Consequently, resuits from different studiesshould not be compared, since conciusions regard-ing the value of one product compared with anotherwould be invaiid. Meta-analysis was unfortunatelyprecluded because of considerabie variability in themethods used in the 144 studies reviewed, and acomparison of the materiais was not possibie.

CONCLUSION

Our literature review shows that we are faced with achoice between standardizing tests, thereby en-abiing meaningfui comparisons, or accepting thatunstandardized tests are of limited use.

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304 The Journal of Adhesive Dentistry

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