Long-term nanomechanical properties of biomodified dentin-resin interface components

Abstract

Failures of dental composite restorative procedures are largely attributed to the degradation of dentin-resin interface components. The biomodification of dentin using bioactive agents may improve the quality and durability of the dentin-resin bonds. The aim of this study was to nanomechanically assess the reduced modulus of elasticity (Er) and nanohardness (H) of major components of the dentin-resin interface (hybrid layer, adhesive layer and underlying dentin) biomodified by collagen cross-linkers at 24hrs, 3 and 6 month 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 months and 6 months when compared to a control. The mechanical properties of the adhesive layer and hybrid layer decreased overtime. The biomodification of the dentin-resin interface structures using GD and GSE can increase the mechanical properties of the interface overtime and may contribute to the long-term quality of adhesive restorations.

1. INTRODUCTION

One of the current procedures to restore lost dental hard tissues is the use of adhesive resin composite systems. The adhesion to dentin, the bulk structure of tooth, takes place via hybrid layer formation between resin monomers from the adhesive system and exposed dentin matrix. Thus, the dentin-resin restorative interface is a complex structure involving the interaction of polymers, collagen, non-collagenous protein and minerals. The lack of complete infiltration of resin monomers into the collagen-rich demineralized dentin surface contributes to degradation of the dentin-resin bonds (Hashimoto et al., 2003; Zou et al., 2010). The deterioration of collagen fibrils within the hybrid layer suggests exposed collagen fibrils after the bonding procedure (Breschi et al., 2003). Decreased bond 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 bonded interface.

Collagen cross-linkers increase the denaturation temperature and improve the mechanical properties of collagen based tissues (Han et al., 2003; Sung et al., 2003). Glutaraldehyde (GD) is widely used as a fixative of biological tissues and increases the mechanical properties of various biological tissues (Rivera and Yamauchi, 1993). Nowadays, there is an increased interest in proanthocyanidin from grape seeds extracts (GSE) due to its wide source availability (Sung et al., 1999) and potential use in several health fields. Proanthocyanidins (PA) can be extracted from several fruits, vegetables, nut, seeds, flowers and barks; selective grape seed extracts from Vinis Vitifera grapes have up to 97% PA.

Natural (i.e. PA) and synthetic dentin collagen cross-linkers (i.e. glutaraldehyde) can biomodify the dentin matrix (Bedran-Russo et al., 2010) and enhance the mechanical properties, biodegradation rates and dentin-resin bond strength (Bedran-Russo et al., 2007; Bedran-Russo et al., 2008; Al-Ammar et al., 2009, Macedo et al., 2009). The nano-mechanical characterization of dentin-resin components would enhance understanding of the biomechanics of dentin collagen biomodification of the complex resin-dentin bonds. Limited characterization of the nano-mechanical properties is available for the dentin-resin bonded interface components (Dos Santos et al., 2010).

The aim of this study was to evaluate the effect of two dentin biomodifiers on the long-term nano-mechanical properties of adhesive interface components. The null hypothesis tested was that GD and GSE would not affect the reduced modulus of elasticity and hardness of adhesive layer, hybrid layer and underlying dentin (adjacent to the hybrid layer) dentin after long-term storage (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 at Chicago (protocol # 2006-0229). The teeth were cleaned and kept frozen (−20° C) 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 (3M ESPE. St. Paul, MN, USA) for 15 sec, rinsed for 30 sec, and excess water was removed with absorbent paper. Two consecutive layers of Adper Single Bond Plus (3M ESPE) adhesive system was applied on the surface, air-dried for solvent evaporation and light-cured for 20 sec (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 sec.

Glutaraldehyde (GD)

Restorative procedures were carried out as described above for C group; expect that the dentin surface was immersed in 5% GD (Fisher Scientific) solution at pH 7.2 for one hour (Al-Ammar et al., 2009) and rinsed for 3 minutes prior to adhesive system application.

Grape Seed Extract (GSE)

Restorative procedures were carried out as described above for C group; expect that the dentin surface was immersed in 6.5% grape seed extract (Mega-Natural, Polyphenolics) at pH 7.2 for one hour (Al-Ammar et al., 2009) and rinsed for 3 minutes prior to adhesive system application.

All the samples were kept in distilled water at 37°C for 24 hours following restorative procedure and then sectioned into 2 beams (approximately 1.5 × 1.5 mm thick) using a low speed diamond blade (Isomet 1000, Buehler). The beams were embedded in epoxy resin and allowed to cure for 8 hours. The specimens were polished using #180, 320, 600, 800 and 1200-grit silicon carbide paper (Buehler) and 9, 6, 3, 1 and 0.5µm polycrystalline diamond suspension (Buehler). Samples were stored in HBSS (Hank’s buffered salt solution) at 37°C for the remaining study period. Beams were evaluated at 24hrs, 3 months and 6 months. Re-polishing using diamond suspensions was performed after 3 and 6months storage.

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 hrs, 3 and 6 months after the restorative procedure. A fluid cell Berkovich tip was used at 1000µN load with a standard trapezoidal load function of 5-2-5 seconds. A 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 relationships (Oliver and Parr, 1992):

$$\text{Er}=\mathrm{S}\surd \mathrm{\pi }/2\surd \mathrm{A}$$

Where S is the initial unloading stiffness and A is the projected contact area between the indenter tip and the sample at maximum load.

$$\mathrm{H}=\text{Pmax}/\mathrm{A}$$

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 (p<0.05).

3. RESULTS

The GD and GSE significantly increased the Er and H values of the hybrid layer when compared to control group at 24 hours and 3 month storage (Table 1) (p<0.05). There is no significant difference between the hybrid layer of GD and GSE groups, except for H at 24 hours (p=0.002). The GD group showed higher H of the hybrid layer (0.78 ± 0.25 GPa) when compared to GSE group (0.68 ± 0.25 GPa). After 6 months, there were no statistically significant differences in the H or Er among the groups (p=0.9703 for H and p=0.6688 for Er). H and Er statistically significantly decreased for all groups overtime, especially after 6 months (p<0.05).

Table 1.

Results of the reduced modulus of elasticity (Er) and nano-hardness (H) of the hybrid layer stored for 24hrs, 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	24hr	9.34 (3.19) b A	12.19 (3.18) a A	11.86 (2.97) a A
	3 m	9.42 (2.940 b A	11.28 (3.36) a A	11.23 (4.77) a A
	6 m	7.98 (2.42) a B	7.71 (3.21) a B	7.81 (3.24) a B
H	24 hr	0.46 (0.21) c A	0.78 (0.25) a A	0.68 (0.25) b A
	3 m	0.47 (0.17) b A	0.58 (0.20) a B	0.54 (0.25) a B
	6 m	0.36 (0.11) a B	0.35 (0.13) a C	0.35 (0.20) a C

Lower and upper case letters indicate statistically significant differences (p < 0.05) on each row and column, respectively for Er and H.

Er: reduced modulus of elasticity; H: hardness; GD: glutaraldehyde; GSE: grape seed extract.

The 24 hours data shows that there were no statistically significant differences in the Er or H of the underlying dentin (adjacent to the hybrid layer) among all groups (p=0.075 and p=0.1012, respectively). After 3 and 6 months of storage, GSE treated group showed higher values of Er when compared to control group (p<0.05) (Table 2). At 3 months, GSE showed higher values of Er and H than GD group. There were no differences in the Er and H between GD and GSE after 6 month, whereas both groups showed significantly higher H values than control group (p=0.0019).

Table 2.

Results of the reduced modulus of elasticity (Er) and nano-hardness (H) of the underlying dentin stored for 24hrs, 3 and 6 months in Hank’s solution.

Nano-mechanical properties of the underlying dentin at dentin-resin interfaces – GPa [mean and (standard deviation)]
		Control	GD	GSE
Er	24hr	22.25 ± 3.63 a A	23.78 ± 2.95 a A	22.11 ± 3.54 a A
	3 m	18.44 ± 3.06 b B	19.20 ± 2.73 b B	22.52 ± 3.02 a A
	6 m	12.21 ± 5.90 b C	13.52 ± 5.44 ab C	15.27 ± 4.78 a B
H	24 hr	1.18 ± 0.20 a A	1.10 ± 0.22 a A	1.10 ± 0.26 a A
	3 m	0.85 ± 0.19 c B	0.98 ± 0.16 b B	1.08 ± 0.22 a A
	6 m	0.48 ± 0.29 b C	0.59 ± 0.20 a C	0.65 ± 0.24 a B

Lower and upper case letters indicate statistically significant differences (p < 0.05) on each row and column, respectively for Er and H.

Er: reduced modulus of elasticity; H: hardness; GD: glutaraldehyde; GSE: grape seed extract.

For the adhesive layer, there was a significant decrease in the mechanical properties (Er and H) over time, regardless of the treatment group (p<0.05) (Table 3).

Table 3.

Results of the reduced modulus of elasticity (Er) and nano-hardness (H) of the adhesive layer stored for 24hrs, 3 and 6 months in Hank’s solution.

Nano-mechanical properties of the adhesive layer at dentin-resin interfaces – GPa [mean and (standard deviation)]
		Control	GD	GSE
Er	24hr	3.80 ± 1.36 c A	7.06 ± 0.96 a A	4.87 ± 1.05 b A
	3 m	4.18 ± 0.82 c A	4.62 ± 0.83 b B	5.03 ± 0.99 a A
	6 m	3.33 ± 1.36 a B	3.57 ± 1.13 a C	3.24 ± 0.77 a B
H	24 hr	0.25 ± 0.12 c A	0.50 ± 0.10 a A	0.32 ± 0.14 b B
	3 m	0.24 ± 0.10 b A	0.32 ± 0.09 b B	0.48 ± 0.17 a A
	6 m	0.16 ± 0.07 a B	0.19 ± 0.06 a C	0.17 ± 0.07 a C

Lower and upper case letters indicate statistically significant differences (p < 0.05) on each row and column, respectively for Er and H.

Er: reduced modulus of elasticity; H: hardness; GD: glutaraldehyde; GSE: grape seed extract.

4. DISCUSSION

The ability of two dentin biomodifiers to improve the mechanical properties of hybrid layer and underlying dentin was supported by this study. GD and GSE were able to increase both, the reduced modulus of elasticity and hardness at different evaluation periods, therefore, the null hypothesis should be rejected.

The application of GD prior to the adhesive system improved the mechanical properties of hybrid layer at 24 hours and 3 months following bonding procedure (Table 1). GD also improved the hardness of the underlying dentin following 3 and 6 months of the bonding procedure when compared to the control group (Table 2). The GD has been shown to decrease the rates of tissue degradation (Sung et al., 1999a; Sung et al., 1999b). GD is capable of fixing proteins due to its molecular affinity for active nitrogen groups of amino acids (Munksgaard and Asmussen, 1984; Pashley et al., 2001; Cilli et al., 2009). GD reacts primarily with amino groups of Lys and Hyl residues and a network of exogenous cross-links can be induced intramolecularly and intermolecularly within collagen (Sung et al., 1999a). It can also inhibit root caries (Walter et al., 2008) and prevent root demineralization (Arends et al., 1989; Dijkman et al., 1992). Enhanced mechanical properties have been observed by application of low concentrations of GD on demineralized dentin (Bedran-Russo et al., 2008). Furthermore, GD improved the resin-dentin bonding interfaces (Al-Ammar et al., 2009; Cilli et al., 2009). The greater disadvantage of GD is its high cytotoxicity, therefore a lower concentration product is required (Sung et al., 1999a). To overcome this disadvantage, naturally occurring cross-linkers have been studied, including proanthocyanidin (Bedran-Russo et al., 2007).

A GSE improved the reduced modulus of elasticity and hardness of the hybrid layer when compared to a control group at 24 hours and 3 months storage (Table 1). Underlying dentin of GSE treated samples showed enhanced mechanical properties at 3 and 6 months when compared to control group (Table 2). PA, the main component of the GSE (97.8%, provided by the manufacturer), is a mixture of monomers, oligomers, and polymers used as natural antioxidants and free-radical scavengers (Fujii et al., 2007). PA is known to stabilize and increase the cross-linkage of type-I collagen fibrils (Masquerlier et al., 1981). The primary mechanism of collagen stabilization with PA is the formation of hydrogen bonding between the protein amide carbonyl and the phenolic hydroxyl (Han et al., 2003). Elm cortex, rich in PA, showed inhibitory effects against proteases including metalloproteinases in periodontal disease (Song et al., 2003). The stability of dentin collagen-PA complex most likely contributed to the higher values of hardness and reduced modulus of the hybrid layer and underlying dentin, especially after 3 and 6 months following the bonding procedure. Strengthening of dentin (Bedran-Russo et al., 2007; 2009) and enhanced dentin-resin bond strength (Al-Ammar et al., 2009; Macedo et al., 2009) has been previously observed using micro-scale evaluations. The present study assessed overtime the biomechanical behavior of individual components of the treated bonded interface.

One of the main factors that can influence the clinical longevity of hybrid layer is the degradation process of unprotected collagen fibrils during the bonding procedure (Breschi et al., 2008). Two degradation patterns could be associated including disorganization of collagen fibrils and hydrolysis of the resin component 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 many exposed collagen fibrils within the hybrid layer, especially for total-etch adhesives (Breschi et al., 2008) as the one used in the present study. The present data shows decreased nanomechanical properties of the hybrid layer and underlying dentin over time, especially after 6 month. The use of collagen biomodifers alone does not fully prevent the decrease in the nano-mechanical properties overtime.

There was a significant decrease in the mechanical properties of adhesive layer over time (Table 3). The water storage of the samples could promote a softening of the polymer network by water (Ferracane, 2006). The water sorption into adhesive polymers is related to the hydrophilicity of adhesive, especially in HEMA-containing systems (Hosaka et al., 2010). Single Bond has shown higher values of water sorption and solubility compared with non-solvated systems (Malacarne et al., 2006). Hence, the elastic modulus of Single Bond is affected by long-term storage (Yasuda et al., 2008). The decrease in the hardness and reduced modulus of adhesive layer herein can me explained by the water sorption and plasticization of hydrophilic resins from resin-based materials (Ito et al., 2005).

The present study demonstrates that grape seed extract and glutaraldehyde could improve the mechanical properties of resin-dentin bonded interface components. Enhanced strength and stability of the hybrid layer and underlying dentin by specific tissue biomodifiers may increase long-term durability of the interface. The use of a natural and low cytotoxicity product, such as GSE, seems to be promising during the bonding procedure. Further in vitro and in vivo studies are necessary to evaluate the durability of bonding procedure with biomodification of dentin matrix using clinically relevant application times or using slow delivery systems of the agent.