Effects of solvents on the early stage stiffeningrate of demineralized dentin matrix

Fernanda C.P. Garciaa, Masayuki Otsukib, David H. Pashleyc,Franklin R. Tayd, Ricardo M. Carvalhoa,*

aDepartment of Operative Dentistry, Endodontics and Dental Materials,Bauru School of Dentistry, University of Sao Paulo, Sao Paulo, BrazilbDepartment of Restorative Sciences, Tokyo Medical and Dental University, JapancDepartment of Oral Biology and Maxilofacial Pathology, School of Dentistry,Medical College of Georgia, Augusta, GA 30912-1129, USAdDepartment of Pediatric Dentistry, Prince Philip Hospital, School of Dentistry,University of Hong Kong, Hong Kong, China

Received 30 October 2003; received in revised form 17 August 2004; accepted 5 October 2004

03do

SP

KEYWORDSCollagen;Acetone;Ethanol;Methanol;Propanol;HEMA;Air;Modulus of elasticity;Stiffening rate

00-5712/$ - see front matter q 200i:10.1016/j.jdent.2004.10.013

* Corresponding author. Al. Dr Octav, CEP 17012-901, Brazil. Tel.: C55 1E-mail address: ricfob@fob.usp.br

Summary Objectives. To monitor the stiffening rate of demineralized dentinmatrix at the early stages after exposure to different neat solvents.

Methods. Dentin beams approximately 0.8!0.7!8.0 mm were obtained fromhuman third molars. After covering their ends with resin composite, the middleexposed length of 4.0 mm (gauge-length) was demineralized in 0.5 M EDTA (pH 7.0)for 7 days. The specimens were gripped by a testing machine, pre-loaded to 10 gand cyclically stressed in tension to 5% strain, for 30 repeated cycles (total 20 min)at 0.6 mm/min while immersed in water (control). Then, water was replaced byeither 100% acetone, methanol, ethanol, propanol, HEMA or air and the specimenssubjected to the same cyclic protocol. The maximum apparent modulus ofelasticity (EMax) was calculated for every cycle, plotted as a function of time andsubjected to regression analysis. Stiffening rate was calculated as changes in E(min). Regression analysis examined the relationship between E and time for eachsolvent. Data were analyzed by one-way ANOVA and Student–Newman–Keuls test ataZ0.05.

Results. Regression analysis showed that E increased significantly with time in allwater-free solvents (R2Z0.8–0.99). Stiffening rate was higher for acetone(0.9 MPa/min) and ethanol (0.8 MPa/min), intermediate for air (0.7 MPa/min),methanol (0.6 MPa/min) and propanol (0.5 MPa/min), lower for HEMA (0.2 MPa/min)and practically none for water (0.07 MPa/min) with p!0.05.

Conclusions. The solvent-induced stiffening rate of demineralized dentin matrixis both time and solvent-dependent. The ability of solvents to promptly stiffen

Journal of Dentistry (2005) 33, 371–377

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io Pinheiro Brizola, Faculdade de Odontologia de Bauru, Universidade de Sao Paulo, 9-75, Bauru,4 3235 8321; fax: C55 14 3224 1388.

(R.M. Carvalho).

F.C.P. Garcia et al.372

the demineralized dentin matrix may be important in maintaining the resin-infiltrated matrix expanded during the solvent evaporation stage of resin bonding.q 2004 Elsevier Ltd. All rights reserved.

Introduction

One of the major problems in resin–dentin bondingis the reduction in the width of interfibrillar spacesfor resin infiltration in demineralized dentin.Shrinkage of the demineralized dentin matrix canoccur during air-drying, or the application of thebonding agents1–3 or during solvent evaporation.4

When water is removed from the matrix by air-drying, collagen fibrils are brought to closerapproximation and undergo interpeptide hydrogenbonding (H-bond). This causes maximum shrinkageof the matrix.1,3 The strength of collagen-to-collagen H-bonds has been calculated and verifiedexperimentally.2 Conversely, when wet-bonding todentin, removal of water is accomplished by theorganic solvents present in the adhesive system.However, since all organic solvents used in adhesivebonding have lower H-bonding affinities thancollagen H-bonding, replacement of water by neatsolvents causes shrinkage of the dentin matrix.2

Theoretically, when water is replaced by solvents,the amount of shrinkage should be inversely relatedto the solubility parameter of the solvent. That is,the lower the solubility parameter, the greater theshrinkage of the matrix.2 The Hansen’s solubilityparameter of collagen peptide has been estimatedto be approximately 18 (J/cm3)1/2.2 However,regardless of the fact that some solvents have asolubility parameter for H-bond lower than thatestimated for collagen peptides, the amount ofshrinkage caused by such solvents never reach thelevel caused by air-drying.2 This is probably becausethe slower rate of shrinkage caused by solvents2

permits the stiffening of the matrix before it shrinkscompletely.5 This phenomenon is used in preparingsoft tissues for microscopy by passing specimensthrough increasingly concentrated alcohols todehydrate specimens and minimize dimensionalchanges. Although solvents with low solubilityparameters for H-bond are expected to causesignificant shrinkage of the dentin matrix, if theyare capable of stiffening it at a fast rate, the netamount of shrinkage will be reduced and interfi-brillar spaces may be better maintained.

It is known that when demineralized dentin isequilibrated with neat solvents for several minutes,its modulus of elasticity increases significantly.5,6

However, from the clinical bonding perspective,

it is important to determine how fast solventscompete with water for the peptide H-bonding, andhence how fast the matrix stiffens in order toreduce the amount of shrinkage, and maintain thespaces between the collagen fibrils as large aspossible to facilitate resin infiltration.

The purpose of this study was to determine thestiffening rate of demineralized dentin matrix afterimmersion in neat polar solvents. The null hypoth-esis was that there are no differences in thestiffening rate of demineralized dentin caused bydifferent neat solvents.

Materials and methods

Extracted intact human third molars stored in watercontaining a few crystals of thymol at 4 8C wereused for this study. Teeth were collected under aprotocol approved by the Ethics Committee Boardof Bauru School of Dentistry, University of SaoPaulo, Brazil. Rectangular-shaped dentin beams of0.7!0.8!8.0 mm were obtained from mid-coronaldentin of the teeth using a low speed diamond saw(Labcut 1010, Extec, Enfield CT, USA) under waterlubrication (Fig. 1a). Both ends of the dentin beamswere bonded with an adhesive (Single Bond, 3M-ESPE, St Paul, MN, USA) and covered with resincomposite (Z100, 3M-ESPE, St Paul, MN, USA). Acentral length of 4.0 mm was left exposed (gauge-length) (Fig. 1b). The specimens were decalcifiedwith 0.5 M EDTA (pH 7.0) at room temperature(25 8C) with agitation for 7 days. This decalcifica-tion protocol has been shown to be effective incompletely demineralizing the specimens.1,5 Afterdecalcification, specimens were washed with waterfor 24 h and stored in deionized water at 4 8C untiltesting. The length, width and thickness of thedecalcified portion of the specimens weremeasured with a digital micrometer (Digimess,Shinko Precision, China), under a light microscopeto avoid compression of the softened surfaces. Thiswas done while the specimens were immersed in25 8C deionized water to avoid premature dehy-dration of the specimens.

Both ends of the specimen were firmly clampedwith friction grips to a universal testing machine(Vitrodyne, V1000, Chatillon, Greensboro NC, USA).After pre-loading to 10 g, each specimen was

Figure 1 Schematic illustration the preparation of0.7 mm thick disks from mid-coronal dentin; how the ‘I’beams were obtained from the disk, their dimensions andtheir appearance after demineralizing their centralregion.

Figure 2 Schematic illustration of how the specimenswere tested under immersion in the respective solutions.

Stiffening rate of demineralized dentin 373

repeatedly stressed in tension at 0.6 mm/min up to5% strain (approximately 200 mm) for 30 cycles,while immersed in distilled water (total timeZ20 min). The load/displacement data were col-lected during the entire cycling into a computercoupled to the machine. Since the actual strain ofthe specimen was not measured with an extens-ometer, but only inferred from the displacement ofthe crosshead, the specimen stiffness values aretermed apparent moduli of elasticity (E). Immedi-ately after collecting the data from immersion inwater, the water was replaced by one of thefollowing neat solvents: acetone (MERCK, S.A., Riode Janeiro, RJ, Brazil), methanol (QUIMISLABOR,Marılia, SP, Brazil), ethanol (LABSYNHT, Diadema,SP, Brazil), propanol (LABSYNHT, Diadema, SP,Brazil) or HEMA (SIGMA CHEMICAL CO., St Louis,MO, USA), and immediately subjected to the samecycling protocol. These solvents were used as theywere received from the manufacturers, without anyadditional treatment. Fresh containers wereopened each working day to avoid contaminationby atmospheric moisture. Five specimens were usedin a randomized order in such a way that all thespecimens were subjected to all the solvents. Aftertested in each solvent, the specimen was immersedin 500 ml of water for 160 min with continuousstirring until completing the series with the otherspecimens. This permitted extraction of residual

solvent from each specimen from the previous testand complete rehydration of the matrix. At the endof these trials the specimens were allowed to dry inair (23G1 8C, 45% RH) while clamped in themachine, and the same cyclic, low-strain apparentmodulus of elasticity was measured for 20 min. Theload/displacement curves were transformed tostress/strain curves and the apparent maximummodulus of elasticity at 5% strain (Emax) wascalculated for every cycle. Regression analysis wasused to demonstrate changes in the Emax as afunction of time. Stiffening rate was calculated aschanges in E (min) (Fig. 2). The stiffening slopeswere compared by a one-way ANOVA to determineif there were any significance differences amongthe groups. Multiple comparisons were done toisolate and compare the significant results usingStudent–Newman–Keuls test at aZ0.05. Regressionanalysis examined the relationship between E andtime for each solvent.

Results

The effects of solvents on the stiffening rate andapparent modulus of elasticity (E) of demineralizeddentin specimens are summarized in Table 1 andFig. 3. Regression analysis showed that E increasedsignificantly with time in all water-free solvents(R2Z0.8–0.99, p!0.05). Stiffening rate was higherfor acetone and ethanol, intermediate for air,methanol and propanol, and lowest for HEMA.Specimens immersed in water showed no changesin the E over time (Table 1). Typical Emax/timecurves are presented in Fig. 3. At cycle 1 (40 s), no

Table 1 The maximum apparent modulus of elasticity (E Max) at representative cycles and stiffening rate(MPa/min) of demineralized dentin in response to solvents.

Solvents Stiffening rate(MPa/min)

EMax (MPa)a

Cycle 1 Cycle 15 Cycle 30

Water K0.07G0.03d 10.9G2.5a 10.0G2.6a 9.4G2.3aHEMA 0.21G0.15cd 12.3G8.3a 13.4G8.6ad 15.7G11.0aPropanol 0.54G0.19ab 18.9G10.4a 24.6G10.9bc 30.1G13.1bcMethanol 0.59G0.17ab 21.3G4.2a 29.2G6.8bc 33.3G7.1bcAir 0.68G0.44ab 16.4G7.9a 18.6G7.9ac 28.7G12.6bcEthanol 0.80G0.27ab 15.5G5.8a 24.8G8.9bcd 31.7G11.0bcAcetone 0.93G0.24ab 18.5G3.4a 28.4G4.3bc 37.4G6.1bc

E-values are in MPa G SD, NZ5 specimens in each group, Mean values designated with the same letter are not statistically differentp!0.05 (per column).

a 5% Strain apparent modulus of elasticity.

F.C.P. Garcia et al.374

significant changes in the Emax were observed for allsolutions (pO0.05), except for methanol thatcaused significant increase in the Emax as comparedto water (p!0.05). Table 2 indicates the signifi-cance among the Emax values for all the solutionsfrom cycles 2 to 7 (approximately 40 s/cycle). After10 min, specimens immersed in acetone, ethanol,methanol and propanol were significantly stifferthan specimens subjected to water or HEMA and nodifferent from air (p!0.05). The Emax of specimensimmersed in water did not change significantly (pO0.05) during all the cycling period, and wassignificantly lower than that resultant from immer-sion of the specimens in all other solvents at the endof the cycling protocol (20 min, p!0.05).

Discussion

It is generally accepted that the primary bondingmechanism of adhesive systems is obtained by

Figure 3 Changes in the average EMax (MPa) ofdemineralized dentin as a function of immersion time(min) in neat solvents. Water served as the control andinduced no changes in E.

hybrid layer formation into acid-etched dentin.7–9

Many factors may interfere with this event.Acid etching of dentin removes its stiff mineralcomponent and results in significant reductions inits mechanical properties.10 For instance, themodulus of elasticity and the ultimate tensilestrength of dentin drop significantly after deminer-alization.11 Such drastic reduction in the mechan-ical properties of dentin is, however, dependent onthe hydration condition of the substrate duringtesting. If demineralized dentin is tested while fullyhydrated, water within the collagen fibrils act as aplasticizer and facilitates molecular slippage,thereby lowering its mechanical properties.5,6,11

However, when demineralized dentin is air-dried,the water that replaced the mineral content afteracid etching is quickly evaporated. During evapor-ation, the surface tension between air and watergenerates forces that act to collapse the collagenfibrils down to their minimal volume.1 Oncecollagen fibrils are brought close together, theyform intermolecular associations, mainly rep-resented by hydrogen bonding, that tend to stiffen

Table 2 Significance among the EMax average valuesfor all solutions from cycles 2 to 7 (per column). AOBOC at p!0.05.

Solvents Cycle2

Cycle3

Cycle4

Cycle5

Cycle6

Cycle7

Metha-nol

A A A A A A

Acetone AB AB AB AB A AAir AB AB ABC ABC AB ABEthanol AB AB ABC ABC A APropa-nol

AB AB ABC ABC A A

HEMA AB AB BC BC AB ABWater B B C C B B

Stiffening rate of demineralized dentin 375

the dehydrated matrix in a collapsed state.2,5,12 Ifdemineralized dentin becomes stiffer in a collapsedstate, the absence of interfibrillar spaces willinterfere with the infiltration of the resin mono-mers to form the hybrid layer.2,9

The wet bonding technique requires that theorganic solvents used to solubilize the adhesivecomonomers replace the water in the interfibrillarspaces. However, solvents commonly used in thebonding agents have a lower ability than water toform H-bond with collagen peptides, therefore, asthey chemically dehydrate the dentin matrix, theyare unable to completely prevent interpeptideassociation, thereby allowing the matrix to partiallycollapse during adhesive application.3 Dehydrationof the dentin matrix caused by solvents replacingwater also alters the mechanical properties ofdemineralized dentin matrix.5,6 Ideally, the sol-vents should stiffen dentin faster than they cause itto collapse. This would permit the maintenance ofthe interfibrillar spaces for resin infiltration andimprove bond strength.13

Our results showed that all neat solvents pro-duced a gradual stiffening effect on demineralizeddentin matrix within the first 20 min after replacingthe water by solvents. Additionally, the stiffeningrate was different among the neat solvents. Thus,the results require rejection of the null hypothesisthat there are no differences in the stiffening rateof demineralized dentin caused by different neatsolvents. Although the stiffening effect was similarfor all neat solvents and air after 20 min, except forHEMA and the control water (Table 1), methanoland acetone produced faster effects within the firstminutes of immersion (Table 2). This is probablybecause of the lower molecular weight and higherdiffusivity of these solvents relative to the largemolecules that permitted a faster migration intothe specimen with consequent displacement ofwater. No significant stiffening was observedwithin the first 120 s (ca. 3 cycles) for the solvents,except for methanol. Although this indicates thatthe collapse of demineralized dentin cannot beprevented by matrix stiffening within the clinicaltime usually allowed for bonding, such extrapol-ation has to be done with care because ourspecimens of demineralized dentin were muchlarger (ca. 700 mm thick) than the thin deminer-alized layer (ca. 1–5 mm) produced in clinicalbonding. Conversely, if differences in specimensizes could be disregarded, then the results wouldindicate that 120 s is not enough time for displace-ment of a critical amount of water that wouldpermit the solvents to act on the collagen fibrils,except when the methanol was used. Apparently,methanol combines well the ability to maintain

the interfibrillar spaces opened while stiffen thematrix. In a bond strength study where experimen-tal HEMA/solvent primers were applied to air-drieddentin before bonding with a 4-META-TBBO basedadhesive, the HEMA/methanol primer resulted inthe maintenance of the widest interfibrillar spaceswith high bond strength values.13 The high Hansen’ssolubility parameter for H-bonding of methanol maybe able to maintain the collagen scaffold expandedwhile makes it stiffer, thereby preventing it tocollapse when the solvent evaporates.

In the very early stages of water replacement, itis likely a little amount of residual water in themixture is capable of plasticizing the matrix, thuspreventing significant stiffening. Only when thesolvent displaces water below that critical level,will the collagen loses its elasticity and becomesstiff, and that was observed only after 120 s ofexposure to the solvents (acetone and methanol).

As expected, water produced no changes in theE of demineralized dentin. Among the solvents,HEMA produced a significantly lower overall stiffen-ing rate and Emax (p!0.05). This is probablybecause HEMA’s high molecular weight wouldhamper its diffusion within the water filled speci-men. When commercially available adhesive sys-tems are applied to wet dentin, the diffusion ofmonomers such as HEMA into the demineralizeddentin is limited to outer surface layer.14 Althoughnot significant, differences in the stiffening ratecaused by the other neat solvents may be explainedby the ability of the solvents to stabilize thecollagen by increasing the amount of interactionsof weak forces between adjacent collagen mol-ecules and collagen molecules with solvent itself(Van der Waals, ionic interactions, hydrophobicbonds).2 On the other hand, the rate of stiffeningmay be related to how fast the solvent can removewater to permit interpeptide H-bonding. In thiscase, faster stiffening would not necessarily indi-cate that interfibrillar spaces are being preserved,but that they are being rapidly reduced. In terms ofdimensional changes, this would cause an immedi-ate shrinkage of the matrix and latter expansionwould only occur if the solvent has a solubilityparameter for H-bonding that is higher than that ofinterpeptide H-bond.2,15 Indeed, some of ourpreliminary laboratory work on isometric tensiondevelopment of demineralized dentin matrixshowed that the matrix develops immediate shrink-age tension when water is replaced by solvents(Carvalho et al., 1995, unpublished data). A similarphenomenon has been observed when deminera-lized dentin matrix is exposed to acids.16 Thedegree of stiffening caused by dehydration withsolvents seems to be dependent on the respective

F.C.P. Garcia et al.376

solubility parameter for H-bonding (dh) of thesolvent.5 That is, the higher the dh, the lower thedegree of stiffening. This is because solvents withhigher dh preferentially interact with collagenpeptides, thus preventing interpeptide H-bonding,that would generate the highest stiffening effect asin air-dried dentin.5,6 The time-dependence of theincrease in stiffness was probably due to the timenecessary for these solvents to diffuse into thedemineralized dentin matrix and then into thecollagen fibrils, followed by the time necessary forthese hydrogen bonds to form.

In this study, the highest apparent modulus ofelasticity that could be observed for demineralizeddentin collagen was in the range of 10.9 MPa inwater to 37.4 MPa in acetone after 20 min ofimmersion. The values for specimens immersed inwater are very close to previous values obtained byour group (Pashley et al., 2003), however, valuesfor specimens immersed in the solvents or exposedto air (28.7 MPa) were slightly lower than previouslyreported.5 This is because in this study wemeasured E within the first 20 min after exposureto the solvents, while in our previous studies thespecimens were equilibrated in the solvents foreither 30 min6 or 60 min5 before testing. This alsoindicates that stiffening would gradually increasehad the specimens being immersed in the solventsfor longer periods. The initial stiffening is probablycaused by displacement of unbound water that isfreely located within the interfibrillar spaces, andslower, latter stiffening may be related to displace-ment of water bound within the collagen fibrils.Contrary to what could be expected, specimensexposed to air did not show the highest E. It is likelythat the slow rate of water evaporation at a 44% ofrelative humidity did not allow for complete speci-men dehydration during the 20 min testing time,and residual water was still present within thespecimen, thus lowering its stiffness. Indeed,projections from the plotting of changes in E asa function of time (Fig. 3) indicate that the moduluswould increase more had the specimens beingimmersed for longer times in the solvents orexposed to air.

Clinically, mixtures of solvents and monomersare applied for 20–30 s during bonding procedures.Although diffusion distances in that situation aremuch smaller (1–5 mm), it is unlikely that suchapplication times would permit a degree of stiffen-ing capable of preventing collapse, particularlywhen the solvents are evaporated.4 When mixturesof 35% HEMA with 65% methanol or ethanol wereapplied to air-dried dentin for 60 s before bonding,the combination of re-expansion and stiffening ofthe collagen matrix was probably good enough to

permit maintenance of the interfibrillar spaces forresin infiltration that resulted in improved bondstrengths.13 Clearly, if demineralized dentin matrixis to be dehydrated by solvents and the interfibrillarspaces preserved at the same time, then solventswith higher dh values are desirable. The short,clinically acceptable times that are currently usedin resin–dentin bonding may need to be re-exam-ined and optimized.

Conclusion

Water (control) produced no stiffening in thedemineralized matrix. The stiffening rate ofdemineralized dentin matrix is both time-andsolvent-dependent. The anticipated hypothesiswas, therefore, rejected.

Acknowledgements

Supported, in part, by grants: 300481/95-0,474226/03-4 from CNPq; 01/06140-1 and02/06682-1 from FAPESP, Brazil; and by grantDE 014911 from the NIDCR, Bethesda, MD, USA.We are grateful to Mr Kazuo Kato for theillustrations.

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