Journal of Biomechanics 44 (2011) 1691–1694

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Journal of Biomechanics

0021-92

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Long-term nano-mechanical properties of biomodified dentin–resininterface components

Paulo Henrique Dos Santos a,b, Sachin Karol b, Ana Karina Bedran-Russo b,n

a Department of Dental Materials and Prosthodontics, Arac-atuba School of Dentistry, UNESP, Arac-atuba, SP, Brazilb Department of Restorative Dentistry, College of Dentistry, University of Illinois at Chicago—UIC, 801 S Paulina St., Chicago, IL 60612, USA

a r t i c l e i n f o

Article history:

Accepted 26 March 2011Failures of dental composite restorative procedures are largely attributed to the degradation of dentin–

resin interface components. Biomodification of dentin using bioactive agents may improve the quality

Keywords:

Collagen

Adhesion

Mechanical properties

Dentin–resin bonds

nanoindentation

Dental adhesive systems

Proanthocyanidin

90/$ - see front matter & 2011 Elsevier Ltd. A

016/j.jbiomech.2011.03.030

esponding author. Tel.: þ1 312 996 3535; fax

ail address: bedran@uic.edu (A.K. Bedran-Rus

a b s t r a c t

and durability of the dentin–resin bonds. The aim of this study was to nanomechanically assess the

reduced modulus of elasticity (Er) and nano-hardness (H) of major components of the dentin–resin

interface (hybrid layer, adhesive layer and underlying dentin) biomodified by collagen cross-linkers at

24 h, 3 and 6 months following restorative procedure. Demineralized dentin surfaces were biomodified

with 5% glutaraldehyde (GD) or 6.5% grape seed extract (GSE) prior to placement of adhesive systems

and composite resin. Nano-measurements of the interface components in a fluid cell showed that both

agents increased the Er and H of underlying dentin after 3 and 6 months when compared to a control.

The mechanical properties of the adhesive and hybrid layers decreased over time. Biomodification of

the dentin–resin interface structures using GD and GSE can increase the mechanical properties of the

interface over time and may contribute to the long-term quality of adhesive restorations.

& 2011 Elsevier Ltd. All rights reserved.

1. Introduction

One of the current procedures to restore lost dental hardtissues is the use of adhesive resin composite systems. Adhesionto dentin, the bulk structure of tooth, takes place via hybrid layerformation between resin monomers from the adhesive systemand exposed dentin matrix. Thus, the dentin–resin restorativeinterface is a complex structure involving the interaction ofpolymers, collagen, non-collagenous protein and minerals. Lackof complete infiltration of resin monomers into the collagen-richdemineralized dentin surface contributes to degradation of thedentin–resin bonds (Hashimoto et al., 2003; Zou et al., 2010).Deterioration of collagen fibrils within the hybrid layer suggestsexposed collagen fibrils after the bonding procedure. Decreasedbond strength between adhesive systems and dentin after long-term storage (De Munck et al., 2011) and decreased sealing ability(Li et al., 2001) are indicators of the degradation of the bondedinterface.

Collagen cross-linkers increase the denaturation temperatureand improve the mechanical properties of collagen based tissues(Han et al., 2003; Sung et al., 2003). Glutaraldehyde (GD) iswidely used as a fixative of biological tissues and increases themechanical properties of various biological tissues (Rivera andYamauchi, 1993). Nowadays, there is an increased interest inproanthocyanidin from grape seed extracts (GSE) due to its wide

ll rights reserved.

: þ1 312 413 9581.

so).

source availability (Sung et al., 1999a, 1999b) and potential usein several health fields. Proanthocyanidins (PA) can be extractedfrom several fruits, vegetables, nut, seeds, flowers and barks;selective grape seed extracts from Vinis vitifera grapes have up to97% PA.

Natural (i.e. PA) and synthetic dentin collagen cross-linkers (i.e. glutaraldehyde) can biomodify the dentin matrix(Bedran-Russo et al., 2011) and enhance the mechanical proper-ties, biodegradation rates and dentin–resin bond strength(Bedran-Russo et al., 2007, 2008; Xie et al., 2008; Al-Ammaret al., 2009; Macedo et al., 2009). The nano-mechanical charac-terization of dentin–resin components would enhance under-standing of the biomechanics of dentin collagen biomodificationof the complex resin–dentin bonds. Limited characterization ofthe nano-mechanical properties is available for the dentin–resinbonded interface components (Dos Santos et al., in press).

The aim of this study was to evaluate the effect of two dentinbiomodifiers on the long-term nano-mechanical properties ofadhesive interface components. The null hypothesis tested wasthat GD and GSE would not affect the reduced modulus ofelasticity and hardness of adhesive layer, hybrid layer and under-lying dentin (adjacent to the hybrid layer) dentin after long-termstorage (up to 6 months).

2. Methods and materials

In this study nine extracted sound human third molars were used following

approval by the Institute Review Board Committee from the University of Illinois

Table 1Results of the reduced modulus of elasticity (Er) and nano-hardness (H) of the

hybrid layer stored for 24 h, 3 and 6 months in Hank’s solution.

Nano-mechanical properties of the hybrid layer at dentin–resin interfaces(GPa; mean and standard deviation)

Control GD GSE

Er 24 h 9.34 (3.19) bA 12.19 (3.18) aA 11.86 (2.97) aA

3 months 9.42 (2.94) bA 11.28 (3.36) aA 11.23 (4.77) aA

6 months 7.98 (2.42) aB 7.71 (3.21) aB 7.81 (3.24) aB

H 24 h 0.46 (0.21) cA 0.78 (0.25) aA 0.68 (0.25) bA

3 months 0.47 (0.17) bA 0.58 (0.20) aB 0.54 (0.25) aB

6 months 0.36 (0.11) aB 0.35 (0.13) aC 0.35 (0.20) aC

Lower and upper case letters indicate statistically significant differences (po0.05)

on each row and column, respectively, for Er and H. Er: reduced modulus of

elasticity; H: hardness; GD: glutaraldehyde and GSE: grape seed extract.

Table 2Results of the reduced modulus of elasticity (Er) and nano-hardness (H) of the

underlying dentin stored for 24 h, 3 and 6 months in Hank’s solution.

Nano-mechanical properties of the underlying dentin at dentin–resininterfaces (GPa; mean and standard deviation)

Control GD GSE

Er 24 h 22.2573.63 aA 23.7872.95 aA 22.1173.54 aA

3 months 18.4473.06 bB 19.2072.73 bB 22.5273.02 aA

6 months 12.2175.90 bC 13.5275.44 abC 15.2774.78 aB

H 24 h 1.1870.20 aA 1.1070.22 aA 1.1070.26 aA

3 months 0.8570.19 cB 0.9870.16 bB 1.0870.22 aA

6 months 0.4870.29 bC 0.5970.20 aC 0.6570.24 aB

Lower and upper case letters indicate statistically significant differences (po0.05)

on each row and column, respectively, for Er and H. Er: reduced modulus of

elasticity; H: hardness; GD: glutaraldehyde; GSE: grape seed extract.

Table 3Results of the reduced modulus of elasticity (Er) and nano-hardness (H) of the

adhesive layer stored for 24 h, 3 and 6 months in Hank’s solution.

Nano-mechanical properties of the adhesive layer at dentin–resininterfaces (GPa; mean and standard deviation)

Control GD GSE

Er 24 h 3.8071.36 cA 7.0670.96 aA 4.8771.05 bA

P.H. Dos Santos et al. / Journal of Biomechanics 44 (2011) 1691–16941692

at Chicago (protocol #2006-0229). The teeth were cleaned and kept frozen

(�20 1C) for 2–3 weeks. The occlusal surfaces were ground flat with #180, 320

and 600 grit silicon carbide paper (Buehler, Lake Bluff, IL, USA) under running

water to remove enamel and expose dentin surface. The teeth were randomly

divided into three groups according to the restorative procedure:

Control group (C): The dentin surface was etched with 35% phosphoric acid

etchant gel (3 M ESPE, St. Paul, MN, USA) for 15 s, rinsed for 30 s, and excess water

was removed with absorbent paper. Two consecutive layers of Adper Single Bond

Plus (3 M ESPE) adhesive system was applied on the surface, air-dried for solvent

evaporation and light-cured for 20 s (Optilux 501, Kerr Demetron, Danbury, CT,

USA). Premise nanofilled resin composite (Kerr) was built using 3 increments to

form 6-mm-high crowns. Each increment was light-cured for 40 s.

Glutaraldehyde (GD): Restorative procedures were carried out as described

above for C group; except that the dentin surface was immersed in 5% GD (Fisher

Scientific) solution at pH 7.2 for 1 h (Al-Ammar et al., 2009) and rinsed for 3 min

prior to adhesive system application.

Grape Seed Extract (GSE): Restorative procedures were carried out as described

above for C group; except that the dentin surface was immersed in 6.5% grape seed

extract (Mega-Natural, Polyphenolics) at pH 7.2 for 1 h (Al-Ammar et al., 2009) and

rinsed for 3 min prior to adhesive system application.

All the samples were kept in distilled water at 37 1C for 24 h following

restorative procedure and then sectioned into 2 beams (approximately 1.5 mm2

thick) using a low speed diamond blade (Isomet 1000, Buehler). The beams

were embedded in epoxy resin and allowed to cure for 8 h. The specimens were

polished using #180, 320, 600, 800 and 1200-grit silicon carbide paper

(Buehler) and 9, 6, 3, 1 and 0.5 mm polycrystalline diamond suspension

(Buehler). Samples were stored in Hank’s buffered salt solution (HBSS) at

37 1C for the remaining study period. Beams were evaluated at 24 h, 3 months

and 6 months. Re-polishing using diamond suspensions was performed after

3 and 6 months storage.

2.1. Assessment of nano-mechanical properties

The hardness (H) and reduced modulus of elasticity (Er) of dentin, hybrid layer

and adhesive layer were measured using a customized Triboindenter (Hysitron

Inc, Minneapolis, MN) at 24 h, 3 and 6 months after the restorative procedure. A

fluid cell Berkovich tip was used at 1000 mN load with a standard trapezoidal load

function of 5–2–5 s. Trapezoidal load functions ensure that creep does not affect

the modulus calculation. The measurements were performed under hydrated

conditions. Samples were kept immersed in HBSS (Balooch et al., 1998; Habelitz

et al., 2002). Er and H were calculated on the load–displacement curves according

to the following relationship (Oliver and Parr, 1992):

Er¼ Sffiffiffiffi

pp

=2ffiffiffi

Ap

where S is the initial unloading stiffness and A is the projected contact area

between the indenter tip and the sample at maximum load:

H¼ Pmax=A

where Pmax is the maximum load and A is the same projected contact area as

described for the calculation of reduced modulus. In each beam, three indents

were performed in each interface component. The H and Er values of each beam

were calculated by averaging the 3 indents. Statistical analysis was performed

using repeated measurements for ANOVA and Fisher’s PLSD test (po0.05).

3 months 4.1870.82 cA 4.6270.83 bB 5.0370.99 aA

6 months 3.3371.36 aB 3.5771.13 aC 3.2470.77 aB

H 24 h 0.2570.12 cA 0.5070.10 aA 0.3270.14 bB

3 months 0.2470.10 bA 0.3270.09 bB 0.4870.17 aA

6 months 0.1670.07 aB 0.1970.06 aC 0.1770.07 aC

Lower and upper case letters indicate statistically significant differences (po0.05)

on each row and column, respectively, for Er and H. Er: reduced modulus of

elasticity; H: hardness; GD: glutaraldehyde; GSE: grape seed extract.

3. Results

GD and GSE treatment significantly increased the Er and H

values of the hybrid layer when compared to control group at24 h and 3 month storage (Table 1) (po0.05). There is nosignificant difference between the hybrid layer of GD and GSEgroups, except for H at 24 h (p¼0.002). The GD group showedhigher H of the hybrid layer (0.7870.25 GPa) when compared toGSE group (0.6870.25 GPa). After 6 months, there were nostatistically significant differences in the H or Er among thegroups (p¼0.9703 for H and p¼0.6688 for Er). H and Er statisti-cally significantly decreased for all groups over time, especiallyafter 6 months (po0.05).

The 24 h data show that there were no statistically signifi-cant differences in the Er or H of the underlying dentin(adjacent to the hybrid layer) among all groups (p¼0.075 andp¼0.1012, respectively). After 3 and 6 months of storage, GSEtreated group showed higher values of Er when compared tocontrol group (po0.05) (Table 2). At 3 months, GSE showedhigher values of Er and H than GD group. There were no

differences in the Er and H between GD and GSE after 6 months,whereas both groups showed significantly higher H values thancontrol group (p¼0.0019).

For the adhesive layer, there was a significant decrease in themechanical properties (Er and H) over time, regardless of thetreatment group (po0.05) (Table 3).

4. Discussion

The ability of two dentin biomodifiers to improve the mechan-ical properties of hybrid layer and underlying dentin was sup-ported by this study. GD and GSE were able to increase both, the

P.H. Dos Santos et al. / Journal of Biomechanics 44 (2011) 1691–1694 1693

reduced modulus of elasticity and hardness at different evalua-tion periods; therefore, the null hypothesis should be rejected.

The application of GD prior to the adhesive system improvedthe mechanical properties of hybrid layer at 24 h and 3 monthsfollowing bonding procedure (Table 1). GD also improved thehardness of the underlying dentin following 3 and 6 months ofthe bonding procedure when compared to the control group(Table 2). The GD has been shown to decrease the rates of tissuedegradation (Sung et al., 1999a, 1999b). GD is capable of fixingproteins due to its molecular affinity for active nitrogen groupsof amino acids (Munksgaard and Asmussen, 1984; Pashleyet al., 2001; Cilli et al., 2009). GD reacts primarily with aminogroups of Lys and Hyl residues and a network of exogenouscross-links can be induced intramolecularly and intermolecu-larly within collagen (Sung et al., 1999a). It can also inhibit rootcaries (Walter et al., 2008) and prevent root demineralization(Arends et al., 1989; Dijkman et al., 1992). Enhanced mechan-ical properties have been observed by application of lowconcentrations of GD on demineralized dentin (Bedran-Russoet al., 2008). Furthermore, GD improved the resin–dentinbonding interfaces (Al-Ammar et al., 2009; Cilli et al., 2009).The greater disadvantage of GD is its high cytotoxicity; there-fore a lower concentration product is required (Sung et al.,1999a). To overcome this disadvantage, naturally occurringcross-linkers have been studied, including proanthocyanidin(Bedran-Russo et al., 2007).

A GSE improved the reduced modulus of elasticity andhardness of the hybrid layer when compared to the controlgroup at 24 h and 3 months storage (Table 1). Underlyingdentin of GSE treated samples showed enhanced mechanicalproperties at 3 and 6 months when compared to control group(Table 2). PA, the main component of the GSE (97.8%, providedby the manufacturer), is a mixture of monomers, oligomers andpolymers used as natural antioxidants and free-radical scaven-gers (Fujii et al., 2007). PA is known to stabilize and increasethe cross-linkage of type-I collagen fibrils (Masquerlier et al.,1981). The primary mechanism of collagen stabilization withPA is the formation of hydrogen bonding between the proteinamide carbonyl and the phenolic hydroxyl (Han et al., 2003).Elm cortex, rich in PA, showed inhibitory effects againstproteases including metalloproteinases in periodontal disease(Song et al., 2003). The stability of dentin collagen–PA complexmost likely contributed to the higher values of hardness andreduced modulus of the hybrid layer and underlying dentin,especially after 3 and 6 months following the bonding proce-dure. Strengthening of dentin (Bedran-Russo et al., 2007, 2009)and enhanced dentin–resin bond strength (Al-Ammar et al.,2009; Macedo et al., 2009) have been previously observed usingmicro-scale evaluations. The present study assessed over timethe biomechanical behavior of individual components of thetreated bonded interface.

One of the main factors that can influence the clinical long-evity of hybrid layer is the degradation process of unprotectedcollagen fibrils during the bonding procedure (Breschi et al.,2008). Two degradation patterns could be associated includingdisorganization of collagen fibrils and hydrolysis of the resincomponent from interfibrilar spaces within the hybrid layer(Hashimoto et al., 2003). The deterioration of collagen fibrils,detectable both in vitro and in vivo, suggests that there are manyexposed collagen fibrils within the hybrid layer, especially fortotal-etch adhesives (Breschi et al., 2008) as the one used in thepresent study. The present data show decreased nano-mechanicalproperties of the hybrid layer and underlying dentin over time,especially after 6 months. The use of collagen biomodifiers alonedoes not fully prevent the decrease in the nano-mechanicalproperties over time.

There was a significant decrease in the mechanical propertiesof adhesive layer over time (Table 3). The water storage of thesamples could promote a softening of the polymer network bywater (Ferracane, 2006). The water sorption into adhesive poly-mers is related to the hydrophilicity of adhesive, especially inHEMA-containing systems (Hosaka et al., 2010). Single Bond hasshown higher values of water sorption and solubility comparedwith non-solvated systems (Malacarne et al., 2006). Hence, theelastic modulus of Adper Single Bond is affected by long-termstorage (Yasuda et al., 2008). The decrease in the hardness andreduced modulus of adhesive layer herein can be explained by thewater sorption and plasticization of hydrophilic resins from resin-based materials (Ito et al., 2005).

The present study demonstrates that grape seed extract andglutaraldehyde could improve the mechanical properties of resin–dentin bonded interface components. Enhanced strength andstability of the hybrid layer and underlying dentin by specifictissue biomodifiers may increase long-term durability of theinterface. The use of a natural and low cytotoxicity product, suchas GSE, seems to be promising during the bonding procedure.Further in vitro and in vivo studies are necessary to evaluate thedurability of bonding procedure with biomodification of dentinmatrix using clinically relevant application times or using slowdelivery systems of the agent.

Conflict of interest statement

The authors would like to disclose that Dr. Ana Bedran-Russois the inventor of a pending US Patent (#2009/0123581—‘‘Col-lagen cross-linking agents on dental restorative treatment andpreventive dentistry’’) held by the Board of Trustees of theUniversity of Illinois.

Acknowledgments

The study was supported by research grants from Fapesp-Brazil #2008/03213-7 and NIH-NIDCR #DE017740. We are thank-ful to 3M ESPE and Kerr for donation of their dental restorativematerials.

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