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Mechanical stability of resindentin

bond components

Marcela Rocha de Oliveira Carrilhoa, Franklin R. Tayb, David H. Pashleyc,Leo Tjaderhaned,e, Ricardo Marins Carvalhof,*

aDepartment of Restorative Dentistry, Dental Materials, Piracicaba School of Dentistry,

University of Campinas, Piracicaba, SP, BrazilbDepartment of Conservative Dentistry, School of Dentistry, Prince Philip Hospital,

University of Hong Kong, Hong Kong, ChinacDepartment of Oral Biology and Maxillofacial Pathology, School of Dentistry,

Medical College of Georgia, Augusta, GA, USAdInstitute of Dentistry, University of Helsinki, Helsinki, FinlandeDepartment of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, FinlandfDepartment of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry,

University of Sao Paulo, Bauru, SP, Brazil

KEYWORDSResin composite;

Adhesives; Collagen

fibrils; Dentin;

Mechanical properties;

Durability

SummaryObjectives: To evaluate the effects of long-term storage on the mechanicalproperties of the components of resindentin bonds, that is, resin composite, adhesivesystem, demineralized and mineralized dentin.

Methods: Specimens of resin composite (Z250) and adhesive systems (Single Bond-SB; One-Step-OS and Clearfil Liner Bond 2V-CL) were cast in molds. Dentin specimenswere prepared from dentin discs obtained from the crowns of extracted human molars.Specimens of demineralized dentin were obtained by immersion of dentin discs for6 days in 0.5 mol/l EDTA (pH 7.0). Both dentin and resin-based substrates were shapedto hourglass or I-beam specimens that were used to determine the true stress (TS) orapparent modulus of elasticity E;respectively. Control specimens were subjected totensile testing at 0.6 mm/min after 24 h of immersion in distilled water. Experimentalspecimens were stored at 37 8C in either distilled water or mineral oil and testedafter 12 months. The data of each group were individually analyzed by ANOVA andTukeys test.

Results: Both TS and Eof the resin-based materials decreased significantly after12 months of storage in water p , 0:05;except the TS of SB p . 0:05: No changeswere observed for specimens of mineralized dentin, regardless of storage conditionp . 0:05: Storage of demineralized dentin in water did not cause any significanteffect in either TS or E p . 0:05; however, significant reductions of TS and E ofdemineralized dentin occurred after storage in oil for 1 year p , 0:05:

Significance: Storage time and medium may be deleterious to the mechanicalproperties of the resindentin bond components, which ultimately could compromisethe durability of resin dentin bonds.Q 2004 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Dental Materials (2005)21, 232241

www.intl.elsevierhealth.com/journals/dema

0109-5641/$ - see front matter Q 2004 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.dental.2004.06.001

*Corresponding author. Address: Al. Otavio Pinheiro Brisolla 9-75, Bauru, SP, Faculdade de Odontologia de Bauru, Universidade deSao Paulo, Dent

stica, Bauru, CEP 17012-901, Brazil. Tel.: 55-14-235-8321; fax:55-14-224-1388.

E-mail address:[email protected]
http://www.intl.elsevierhealth.com/journals/demahttp://www.intl.elsevierhealth.com/journals/dema


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Introduction

Recent studies revealed that the resin dentinbonds created by current hydrophilic adhesivesystems can severely degrade over time [13].Although several factors may account for suchdeterioration, there has always been a concernregarding how well resin monomers are able toinfiltrate into demineralized dentin, and how thismonomer infiltration might create a stable hybridlayer. The reasons why incomplete infiltration canoccur are numerous, but the end result is a poroushybrid layer[4]highly susceptible to degradation byoral fluids[1,5,6].

Despite our inability to diagnose the early signs

of impending bond degradation [7], contemporarymethods in dental materials investigation havebeen useful to characterize several mechanismsresponsible for detecting either mechanical ormorphological degradation of resin dentin bonds[3,8,9]. Since the mechanical properties of eachresin dentin bond component are expected toplay significant roles in the ultimate bondstrength values [1014], the durability of resindentin bonds depends upon the stability of theircomponents over time. Morphological in vitrostudies indicated that both resin and collagenmatrices may degrade upon storage [6,9]. Cata-strophic failure of resin dentin bonds mayinitiate in one specific component of the inter-face. The identification of which component ismore likely to be responsible for the overallreduction of the bond strength is impossiblewhen the components are evaluated in thecomplex bonded condition. By testing themseparately, the authors expect to be able todetermine which is least stable during variousstorage conditions. Thus, the objective of thisstudy was to investigate the effects of prolongedaging in distilled water or mineral oil on the true

stress (TS) and apparent modulus of elasticity Eof resin dentin bond substrates (i.e. resin com-posite, adhesive system, demineralized andmineralized dentin). Since the materials/sub-strates tested possess viscoelastic behavior andmay undergo deformation upon loading, theultimate cohesive strength at failure is moreproperly named true stress rather than ultimatetensile strength. Similarly, apparent E wasadopted because displacement during testingwas obtained from the crosshead of the machinerather than from extensometers. The null hypoth-esis tested is that there will be no significant

changes in the mechanical properties of thesecomponents, regardless of storage condition.

Material and methods

The institutional review board of the University ofSao Paulo approved this study under protocolnumber 147/00. Fifty-three non-carious humanthird molars were stored in water containing 0.2%sodium azide at 4 8C and used within 6 months ofextraction. Resin composite and adhesive systemsthat were used are presented in Table 1. Theprocedures used to prepare each substrate aredescribed below.

Specimens preparation

Resin compositePolyvinylsiloxane molds with hourglass or I-beamshaped impressions were prepared (Fig. 1). Resincomposite Z250 (3M ESPE, St Paul, MN, USA)approximately 1 mm thick was inserted into eachmold, filling it completely. A glass cover slip wasplaced on the resin composite and the surface waslight-cured with three individual 40 s exposuresusing a light source of 600 mW/cm2, covering theentire surface of the resin composite. Afterpolymerization, cured hourglass or I-beam shapedspecimens were removed from the molds and hand-finished with 600-grit SiC paper. Thirty hourglass

and I-beam shaped specimens were prepared andused to determine the true stress (TS) and apparentmodulus of elasticityE;respectively (Fig. 2). Bothspecimens had a cross-sectional area of 0.7 mm2

(0.1), mean (standard deviation).

Table 1 Resinous materials tested.

Material Composition

Z250 UDMA, Bis-EMA, TEGDMA, inorganic fillerSingle Bond (SB) Adhesive: HEMA, Bis-GMA,

polyalkenoic acid copolymer,

dimethacrylates, ethanol, water and CQOne Step (OS) Adhesive: BPDM, Bis-GMA, HEMA,acetone and CQ

Clearfil LinerBond 2V(CL)

Primer A: MDP,Hydrophilic dimethacrylates, CQ.Primer B: HEMA, N,N-diethanol toluidine,waterAdhesive: MDP, Bis-GMA, HEMA,hydrophobic dimethacrylates,N,N-diethanol toluidine,CQ andcolloidal silica

Bis-GMA: bisphenol A-glycidyl methacrylate; Bis-EMA: bisphe-nol ethoxy methacrylate; BPDM: biphenyl dimethacrylate;CQ: dil-camphorquinone; HEMA: 2-hydroxylethyl methacry-late; MDP: 10-methacryloyloxydecyl dihydrogen phosphate,

TEGDMA: triethylene-glicol-dimethacrylate, UDMA: urethanedimethacrylate.

Durability of resindentin bond components 233


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Adhesive systems

A pilot study showed that adhesive specimensprepared from polyvinylsiloxane molds underwentsevere deformation within a few minutes after

polymerization. To overcome that problem, acrylic

rods were glued to a glass slide to create a15 15 0.8 mm mold to prepare slabs of theadhesives (Fig. 3), as previously described [15].The adhesive systems Single Bond (SB-3M ESPE,St Paul, MN, USA) and One-Step (OS-Bisco, Schaum-burg, IL, USA) were directly dispensed to fill themold completely, left undisturbed for 60 s, andgently blown with oil- and water-free compressedair for an additional 60 s to allow proper solventevaporation. Preliminary testing showed that nofurther weight loss of the adhesives occurredwith prolonged time allowed for evaporation (not

shown). A glass cover slip was placed on theadhesive and the surface was light-cured withfour individual 80 s exposures to a light source of600 mW/cm, covering the entire surface ofadhesive in the matrix.

Equal amounts of primers A and B (sevendrops each) of Clearfil Liner Bond 2V (CL-KurarayMed. Inc., Osaka, Japan) were mixed in a well andimmediately dispensed into the mold. The mixturewas left undisturbed for 60 s and gently air-blownfor an additional 60 s. Then, seven drops of theBond liquid A was added and mixed with theprimers. A glass cover slip was placed on the surface

followed by the same light-curing protocoldescribed above.

Figure 1 Polyvynil siloxane molds used to prepare resincomposite specimens for TS (a) and apparent modulus of

elasticity (b). Z 250 resin composite was directly insertedinto the molds and light-cured.

Figure 2 Representative illustration of the hour-glassshaped specimens used for testing TS and the I beamshaped specimens used for the Etesting.

Figure 3 Schematic of how the adhesive systems weredirectly dispensed into the mold to produce the curedslabs for posterior trimming into hour-glass and I beamshapes.

M.R.O. Carrilho et al.234


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After polymerization, cured slabs measuring15 15 0.8 mm were obtained from the threeadhesives. Each one was sectioned into two halvesand each half was trimmed with a fine diamondbur to either an hourglass or an I-beam shape. Atleast 33 slabs of each adhesive were preparedfrom the matrix, which resulted in 3234 speci-mens for both TS and E: The cross-sectional areaof the specimens for both tests was 0.8 mm2

(^0.05).

Mineralized dentin

Thirty dentin discs (0.8 mm thick) were obtainedfrom the mid-crown of 30 extracted human thirdmolars by means of a diamond saw (Labcut 1010,

Extec Inc., Enfield, CT, USA). The teeth were storedin saline with 0.2% sodium azide and were usedwithin 3 months after extraction. One hourglass andone I-beam shaped specimens were cut from eachdentin disc (Fig. 4) with a fine diamond buroperated in a high-speed handpiece with copiousair-water spray irrigation. This resulted in 30specimens prepared for both TS and E: The cross-sectional area of the specimens for both tests was0.65 mm2 (^0.01).

Demineralized dentin

Twenty-three teeth were used to obtain discs of

mid-coronal dentin. Two hourglass shaped speci-mens were cut from 15 teeth in a similar way asdescribed aboven 30:Both ends of these speci-mens were coated with two layers of nail varnish,leaving a central area of 1.5 mm exposed fordemineralization. Three to four dentin sticks,measuring approximately, 0.80.8 8.0 mmwere obtained from the remaining eight discs, bysimply transversally slicing the discs as describedpreviously[16]. Both ends of the dentin sticks wereacid-etched (35% phosphoric acid, 3M ESPE), rinsed,

blot-dried and coated with two layers of Single Bondadhesive system. Each stick was placed into apolyvinylsiloxane mold with an I-beam shapedimpression and the ends were covered with Z250resin composite, leaving an intermediate area of4.0 mm in length (gauge length) exposed for

demineralization (Fig. 5). Thirty specimens wereprepared. Both the TS (hourglass) and E (I-beam)specimens were demineralized by immersion in0.5 mol/l EDTA (pH 7.0) for 6 days at 25 8C [17]followed by extensive rinsing in water (ca. 24 h).The cross-sectional area of the specimens for TSandEtests was 0.65 mm2 (^0.03).

Storage conditions

Specimens of each substrate/material were dividedinto three groups and randomly assigned to bestored according to the following conditions: 24 hin distilled water (control) and 1 year either indistilled water (1yr W) or in mineral mineral oil

Figure 4 Schematic of how the dentin disc was obtainedfrom the molar teeth and the specimens trimmed out ofthe discs.

Figure 5 Preparation of the dentin beam specimens fordemineralization in EDTA and posterior testing forcalculating E: The dentin beams were placed in thecenter of the mold (a) and the ends covered with resincomposite (b).



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(1yr O), without the addition of preservatives orantimicrobial agents. Storage in mineral oil wasselected to avoid the effects of water storage. Allspecimens were stored in hermetically sealed vialsat 37 8C.

Control specimens testing

The TS and E of each control group were deter-mined after the specimens had been stored for 24 hin distilled water. The specimens were individuallyattached to the grips of a Vitrodyne V-1000(Chatillon Bros., Greensboro, NC, USA) testingmachine and tested in tension at 0.6 mm/min.Specimens for TS (hourglass) were glued to the

testing jig with cyanoacrylate cement (Zapit,Dental Ventures of America, Corona, CA, USA),while E (I-beam) specimens were clamped withspecial grips that permitted the maintenance oftheir 4 mm gauge length. The EDTA-demineralizeddentin specimens were tested while immersed indistilled water. The load-displacement data werecollected in a computer connected to the testingmachine. After failure, the specimens wereremoved from the grips and their cross-sectionalarea measured with a digital caliper to the nearest0.01 mm. The cross-sectional area was used tocalculate the TS as a function of the maximum load

at fracture, and the loaddisplacement data wereconverted to stress strain curves. From thesecurves, the moduli were calculated at the steepestpart of the curve. All values were expressed in MPa.Because displacement was obtained from move-ment of the cross-head instead of an extensometer,the moduli of elasticity have been designated asapparent moduli of elasticity.

Experimental specimens testing

The remaining specimens stored for 1 year wereremoved from the vials, washed in water for 1 h and

tested under the same protocol described above forcontrol specimens.

Statistical analysis

The statistical analysis was not designed to com-pare different substrates. Thus, the data from resincomposite, mineralized dentin and demineralizeddentin substrates were individually analyzed byone-way ANOVA, with the storage condition (24 h,1yr W and 1yr O) as the main factor. Data from theadhesive systems were analyzed by two-wayANOVA, with storage condition (24 h, 1yr W and1yr O) and adhesive systems (SB, OS and CL) as thetwo factors in the analysis. All post hoc multiple

comparisons were performed using Tukeys test.Statistical significance was preset at a 0:05:

Results

Results are summarized in Tables 24. Storage inwater for 1 year did not cause any significant effectin either TS or Eof mineralized and demineralizeddentin specimensp . 0:05:Conversely, storage inwater for 1 year caused significant reductions inboth TS and Eof all resin-based substrates (resincomposite and adhesives) p , 0:05;except for TSof CL adhesive that did not significantly change over

time p . 0:

05:

Reductions in the mechanicalproperties of the self-etch adhesive after 1 yearof storage in water were lower (CL, 29 and 14%,respectively, for Eand TS,Table 4). The mechan-ical properties of both total-etch adhesive resins(SB and OS) were significantly higher at 24 h thanthe self-etch system (CL) p , 0:05: The sametrend was observed after 1 year of water storage,except that TS of OS was not significantly differentfrom that of CL p . 0:05:Storage in mineral oil for1 year did not significantly alter the TS and E ofresin composite (Z250) and mineralized dentinsubstrates, but increased the TS of all adhesive-

substrates (SB, OS and CL) and the Eof OS and CLsignificantly after 1 year of storage in mineraloil p , 0:05: Storage in oil for 1 year caused

Table 2 Changes in the true stress (TS) of each substrate asa function of storage condition.

Substrate Storage condition TS (SD) n

Z250 Control (24 h) 79.7 (23.5) [10]*1 yr W 55.3 (14.2) [10]1 yr O 70.4 (22.7) [10]*

SB Control (24 h) 22.3 (3.3) [13] c,d1 yr W 12.6 (1.9) [10] e

1 yr O 44.3 (6.6) [10] aOS Control (24 h) 18.6 (4.4) [12] d1 yr W 6.8 (1.8) [10] f1 yr O 34.6 (8.9) [10] b

CL Control (24 h) 8.4 (1.5)[13]*f1 yr W 7.2 (1.2) [10]*f1 yr O 25.4 (5.0) [10] c

Mineralized dentin Control (24 h) 82.7 (26.2) [10]*1 yr W 82.7 (15.1) [10]*1 yr O 81.3 (16.8) [10]*

Demineralized dentin Control (24 h) 13.3 (3.6) [10]*1 yr W 12.0 (3.7) [10]*1 yr O 2.4 (0.9) [10]

Values are in MPa (SD) n: (*) Indicates no statisticaldifference among values within each substrate group p .

0:05: Same letter indicates no statistical difference amongadhesive system substrates p . 0:05:



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significant reductions in both TS and Eof deminer-alized dentin specimens p , 0:05:

Discussion

The results of this study showed that the mechan-ical properties of some of the components of theresindentin bonds were significantly affected bystorage in water or oil for 1 year. The nullhypothesis tested must, therefore, be partiallyrejected. The reduction in the mechanical proper-ties of the adhesive systems aged in distilled waterfor 1 year extends and confirms our previousobservations that demonstrated significant

decreases in both TS and Eof the same adhesivesstored in distilled water for 3 and 6 months [15].Cumulative reductions in TS for SB and OS after1-year storage in water reached values of 43 and 63%

of the 24 h controls, while reductions in Ewere inthe range of 35 and 46%, respectively (Table 4). Themechanical properties (ca. TS and E) of CL did notchange significantly when the specimens werestored in water for 36 months[15]. However, thepresent data demonstrate that while no significantchanges were observed for TS, a significantdecrease in the Eof CL was observed after 1-yearstorage. The mechanical properties of CL werealways significantly lower than the other twoadhesive systems tested. The mechanical proper-ties of CL at 24 h were particularly low, and thatmay have accounted for the lack of differencesbetween the control (24 h) and 3 and 6 monthstesting periods [15]. However, the prolonged

immersion time in water may have allowed forfurther deterioration of the polymer structure,thereby resulting in further reductions in itsproperties. It is speculated that between 24 h and6 months, plasticization of the polymer due toimmediate water sorption that occurred within theinitial 24 h of water immersion, was mainly respon-sible for the unaltered properties up to 6 months ofstorage. However, during the following 6 months ofwater storage (up to 1 year), hydrolytic degradationmay have taken place, significantly compromisingthe structure of the polymer. Regardless of thedifferent behavior among the adhesives, the pre-

sent data confirm the cumulative, deleteriouseffects of water to adhesive polymers[8].

Likewise, both mechanical properties of resincomposite specimens (Z250) were also significantlyreduced after 1 year of storage in water p , 0:05:Such changes represented a decrease of 28% in Eand30%inTS(Table 4). Previous studies have shown thatreduction in mechanical properties of resin compo-sites aged in water may occur within 26 months[1820]. Although the authors have not determinedthe mechanical properties of the resin composite inthose periods, the observed reduction of its proper-ties after 1 year is probably a result of a continuous

action of water on the structure of the material. Themechanism of water transport and its effects on themechanical properties of polymers are dependenton several factors[21,22]. Composition and mono-mer ratio varies according to the specific appli-cations and manufacturers goals [23], and suchvariability will define the chemical stability/degrad-ability of resins in a specific environment[8]. It wasdemonstrated that increasing the ratio of TEGDMAto Bis-GMA in a polymethacrylate blend caused anincrease in water uptake[24,25]. Similarly, UDMA-based resin was reported to be highly susceptible towater sorption [26]. Perhaps, the presence ofTEGDMA and UDMA in Z250 resin composite con-tributed to the acceleration of water sorption

Table3 Changes in the apparent modulus of elasticityEofeach resindentin component as a function of storage

condition.Substrate Storage condition E(SD) n

Z250 Control (24 h) 6532 (2136) [10]*1 yr W 4662 (1067) [10]1 yr O 7701 (2199) [10]*

SB Control (24 h) 614 (152) [12]*a,b1 yr W 398 (47) [10] c1 yr O 579 (39) [10]*b

OS Control (24 h) 423 (47) [10] c1 yr W 227 (44) [10] d1 yr O 532 (84) [10] b

CL Control (24 h) 139 (43) [12] e1 yr W 98 (20) [10] f1 yr O 698 (93) [10] a

Mineralized dentin Control (24 h) 7902 (2503) [10]*1 yr W 9046 (1728) [10]*1 yr O 8864 (1987) [10]*

Demineralized dentin Control (24 h) 60 (17) [10]*1 yr W 69 (36) [10]*1 yr O 31 (7.2) [10]

Values are in MPa (SD) n: (*) Indicates no statisticaldifference among values within each substrate group p .0:05: Same letter indicates no statistical difference amongadhesive system substratesp . 0:05:

Table 4 Reduction (%) of the mechanical properties ofresin-based substrates stored in water for 1 year, as afunction of control condition (24 h).

Substrate TS (%) E(%)

Z250 30 28SB 43 35OS 63 46CL 14 29



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and favored balance between water sorption andleaching of resin components. However, during aprolonged soaking this balance can be often upset[21]or cannot actually be achieved for years[27].

Besides the hydrophilic nature of differentmonomers constituting the resin matrix [28],sensitivity of resin-based materials to water effectsalso depends on the degree of polymer cross-linking[21], degree of monomer conversion[29], presenceof fillers, volume fraction of intrinsic nanometer-sized pores [21] and additional environmentalaspects, such as the presence of enzymes andchanges in pH [8,30,31]. Such factors can alsoinfluence the rate of water sorption [3135], and,therefore, can regulate the water effect. Accord-

ingly, in a time-dependent manner, water mol-ecules diffuse into the resin and while some of thesemolecules occupy polymer free volume, othersdisrupt interchain hydrogen bonds, thus alteringthe mechanical properties of the materials [27]. Incontrast, the maintenance or slight increase in Eand TS of all adhesives and resin composite speci-mens aged in mineral oil support the degradingeffects of water storage. The mechanical proper-ties of hydrophilic polymers may be affected byimmersion in water immediately after polymeriz-ation due to a plasticization effect [36]. Conver-sely, if polymers are cured and not exposed to

water, their mechanical properties do not decreaseand even increase over time [15,36]. The resultsobtained with resin-based specimens stored in oil(Table 4) are probably due to the absence of waterin the medium. The higher values observed after1 year of immersion in oil may not be a result ofimproved mechanical properties in this medium,rather, the control values are lower because thespecimens may have suffered the immediate effectsof water immersion after curing. However, wecannot rule out the possibility that long-termimmersion in oil could have dehydrated the speci-mens. This would also explain the higher values

observed after long-term immersion in oil[15].Water storage did not significantly alter the

mechanical properties of EDTA-demineralized den-tin specimens aged for 1 year. These results are inagreement with another study, wherein no signifi-cant changes in either TS or E of EDTA-deminer-alized dentin beams were observed after 48 monthsof storage in buffered saline [17]. Conversely,analysis of fractured interfaces of long-termresindentin bond strength studies have providedevidences that collagen fibrils may degrade overtime [3,37,38]. Phosphoric acid-exposed collagenfibrils unprotected by resin have shown to bemorphologically altered after 500 days of waterstorage[9]. Caries-related literature has suggested

that host-derived proteolytic enzymes that arepresent in saliva and released from demineralizeddentin extracellular matrix can play an importantrole in collagen degradation in caries lesions[39,40]. These endopeptidases, called matrixmetalloproteinases (MMPs), are able to degrademost extracellular matrix proteins, including differ-ent collagens in native and denatured forms [41].The specimens in this study were completelydemineralized in EDTA and thoroughly rinsed withwater before being tested and stored. Even thoughEDTA demineralization removes most dentin MMPs,matrix bound MMPs remain in the demineralizeddentin [42]. However, mineral cations, especiallyZn2 and Ca2 are required to activate MMPs. It is

likely that the MMPs remaining bound to collagenwere not activated due to the absence of metal ionsin the distilled water.

It was noticed that EDTA-demineralized speci-mens exhibited significant reductions in both TS andEafter 1 year of storage in mineral oil. Upon visualinspection, the specimens were seen to be twisted,shrunken and clearly dehydrated (ca. stiff) after1 year immersed in oil. When they were re-hydratedby re-immersion in water just before being tested,they immediately became less twisted and softerthan specimens that were stored in water for thesame period. The softness and weakness of the

specimens were then confirmed by the dramaticloss of their mechanical properties. Any residualcalcium ions that were slowly released from themineralized ends of the specimens bonded withresin composite may have been diluted by the largevolume of water to levels that were so low that theMMPs remained inactive. Conversely, we mayspeculate that when the specimens were incubatedin oil, the water that remained in the matrix wasconfined in situ by the oil. Any calcium releasedfrom the mineralized ends of the specimensremained in this residual water and could haveactivated the MMPs, allowing them to degrade the

matrix over time. However, we believe that is notthe case. The dehydrated appearance of the speci-mens when removed from the oil indicated thatremaining water diffused out from the matrix, thuspreventing the diffusion of metal ions from thedemineralized ends throughout the specimen.Additionally, the specimens in this condition invari-ably ruptured in the middle of the 4 mm exposedarea. If the above rationale was correct, failureswere more likely to occur near the mineralized endsof the specimens. While the possibility that the oilitself may have somehow damaged the collagenmatrix over 1 year storage cannot be ruled out, tothe extent of the authors knowledge, there is noinformation available on the effects of long-term



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dehydration on the mechanical properties of EDTA-demineralized dentin matrix. Both aspects warrantfurther investigation.

No changes in TS or E were observed formineralized dentin specimens aged either inwater or mineral oil during 1 year. It has beenreported that the ultimate tensile strength (UTS,more properly named true stress (TS, in thisstudy) is in the range of 70100 MPa, dependingon the tubule orientation, specimen shape andsize [10,4345]. Values of TS for mineralizeddentin in this study fit well within those valuesfor specimens of similar shape and size. Vari-ations in the UTS or TS values of mineralizeddentin specimens are likely to be regulated by

flaw distribution in the specimen [46]. As thereare no reports indicating that mineralized dentinmechanically degrades over time, therefore, ourexpectations of no changes after storage wereconfirmed. Conversely, the lack of changes in theE deserves further discussion. Recent reportsindicate that the modulus of elasticity of miner-alized dentin may significantly decrease aftershort-term (ca. 5 15 days) storage in water[46,47]. Those studies used a modified atomicforce microscope indentation technique that iscapable of determining the mechanical propertiesof the near-surface layer of the specimens. In

that case, storage in water caused partialdemineralization of the surface layer, and thatwas detected by the surface sensitive method. Itis likely that specimens stored in water in thisstudy also suffered the surface demineralizationeffect of water, however, that was probablyinsignificant enough to cause an overall change inthe bulk specimen. The authors observed valuesof Eof mineralized dentin are below the claimedtrue value of modulus of elasticity for miner-alized dentin (ca. 1929 GPa)[46]. Smaller valuesof modulus of elasticity may be determined by astrain-rate-dependent viscoelastic response, non-

uniform stress distribution that occurs in smallspecimens or flaws introduced during specimenpreparation [46]. The specimens in the currentstudy were small in size and a low strain ratewas used. Moreover, since an extensometerwas not used, displacement was directlymeasured from the cross-head movement ofthe testing machine, therefore, these valuesshould be regarded as apparent modulus ofelasticity. Although the absolute values in thepresent study differ from literature values, themaintenance of E values over time in oil areremarkable.

It would be desirable to apply pretreatments oruse modified materials that would maintain

the stability of resindentin bond components invivo. This would prevent replacements of restor-ations due to degradation over time. Currentbonding techniques and materials have not yetproved to be stable over time in clinical service.Contemporary resin components (adhesives andresin composite) are intrinsically susceptible towater sorption. Ideally, bonding procedures shouldbe done in the absence of water, employinghydrophobic resins that are expected to be morestable over the long-term. At the same time, thesebonding procedures should be capable of inactivat-ing matrix derived proteolytic enzymes. Lowconcentration of chlorhexidine (0.03%), a potentantimicrobial agent, has been shown to hinder the

activity of several MMPs [48], but its effects onresin dentin bonds formation and durability needsto be investigated. For instance, Tetracyclinehydrochloride and EDTA, both used in root surfaceconditioning in periodontal surgery [49], and bothknown to inhibit MMPs[50]might provide means tocontrol the activity of dentin-bound MMPs if used asconditioning agents in resindentin bonds. Futurestudies should focus on long-term experiments thatcan validate whether the durability of resindentinbonds can be improved by using either hydrophobicmonomers and/or protease-inhibitors.

Acknowledgements

The authors wish to thank Mr Eduardo RochaPansica for illustrations. This study was supported,in part, by grants: 99/10043-0, 02/06682-1 and01/07250-5 from FAPESP, 300481/95-0 and474226/03-4 from CNPq, DE014911 from NIDCR,and 104337 from the Academy of Finland.

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