Citation

Shi-Qiang Gong, Zhen-Jie Xue, Shu-Teng Liao, Ya-Bo Wu, Yan Liu. The effect of NDGA-modified etchant on the enzymatic degradation resistance and mechanical properties of collagen matrix[J]. Chin. Chem. Lett., 2018, 29(1): 205-208  

Shi-Qiang Gong^a,1, Zhen-Jie Xue^b,1, Shu-Teng Liao^a, Ya-Bo Wu^a, Yan Liu^c    

Type Ⅰ collagen is the major component of connective tissues such as skin, tendons, bone, and dentin. In the field of tissue engineering and regeneration, collagen has been extensively used as the main component of scaffold that provides hierarchical compartments for cells [1-10]. However, dentin regeneration remains challenging, as it does not remodel [11, 12]. Resin-dentin bonded interface has been considered a unique form of tissue engineering in which a collagen matrix scaffold is reinforced by resin to produce a hybrid layer that couples resinous material to the underlying intact dentin. However, contrary to firm and stable resin-enamel bonding, the durability of resin-dentin bonding is often unsatisfactory. In 1982, Nakabayashi et al. first proposed that hybrid layer formed by the entanglement between collagen fibrils and adhesive resin is the basis for resin-dentin bonding [13]. Since hybrid layer is the mixture of dentin matrix, residual hydroxyapatite, resin monomer and adhesive [14], degradation of any component will affect the stability of hybrid layer, resulting in the failure of dentin bonding. Incomplete penetration of the resin adhesive into the demineralized collagen matrix makes the matrix exposed, and the unprotected collagen is vulnerable to degradation by endogenous and exogenous enzymes and hydrolysis. A large number of studies have shown that the destruction of dentin collagen in the mixed layer is an important reason for the failure of dentin bonding over time [15]. Therefore, taking certain measures to enhance the stability of collagen matrix in the hybrid layer and resist the damage caused by various internal and external factors has become an effective way to improve the durability of dentin bonding.

Introducing exogenous crosslinks to dentin matrix is a stable approach to enhance the biostability of collagen through improving its mechanical properties and resistance to collagenase degradation. Khor et al. have pointed out the significance of the intramolecular and intermolecular chemical bonds of collagen molecules to the stability of collagen fibrils [16]. The chemical crosslinking agents, which increase the number of intramolecular and intermolecular chemical bonds, can effectively increase the stability of collagen structure. Meanwhile, crosslinking may inactivate endogenous enzymes by lowering the molecular mobility of the catalytic sites, which are critical for their protease activities [17, 18]. Traditional chemical crosslinking agents such as glutaraldehyde (GA) and carbodiimide can effectively crosslink collagen matrix in a relatively short period of time. GA is a monomer containing five carbon atoms of aliphatic molecules, and two aldehyde groups at both ends of the hydrocarbon chain. GA crosslinks collagen matrix through chemical bonds between two aldehyde groups and amino groups of collagen molecule [19]. However, one of the unavoidable problems with these chemical cross-linking agents is their potential cytotoxicity, which greatly limits their clinical use. Therefore, significant efforts have been devoted to seek for effective and safe naturally occurring protein crosslinking agents [20-24]. Nordihydroguaiaretic acid (NDGA), isolated from the creosote bush, is a natural di-catechol antioxidant, with well-known anti-inflammatory, anti-cancer and anti-cardiovascular disease properties. The structure of NDGA molecule is simple, and contains two o-catechol groups at the ends of its short alkyl chain. Recent studies have also identified its highly efficient protein crosslinking effect [25, 26]. Although NDGA have good stability of collagen crosslinking, when applied to clinical dental adhesive, its strong free radical scavenging effect will seriously affect the polymerization of resin [27]. Liu et al.[28] put forward for the first time to make a composite etchant by adding proanthocyanidins (PA) into phosphoric acid (PhA), and found that the composite etchant could be used as a dentin etchant and crosslinking agent at the same time. However, relatively large molecular size of PA may adversely influence the validity of PA as a collagen matrix bio-modifier [29].

The purpose of current study was to validate the concept of etch-and-crosslink technique by adding collagen crosslinker into PhA to produce a bio-modified etchant with collagen-stabilizing capability (Scheme 1). In this study, 0.5 wt% or 1 wt% collagen crosslinkers (NDGA or PA) were added into PhA, with dimethyl sulfoxide (DMSO) as co-solvent. Dentin collagen matrix that were demineralized by NDGA or PA-modified etchants were characterized in terms of enzymatic degradation resistance and mechanical properties (Supporting information).

Scheme 1

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Scheme 1. Schematic illustration of etch-and-crosslink technique by using NDGA-modified etchant.

Experimental details can be found in Supporting information.

When demineralized dentin collagen was challenged by collagenase, collagenases approach to collagen molecules and unwind triple helical conformation in such a way that the catalytic active can attack the specific glycine-isoleucine peptide bonds and cleave individual chains in succession [30]. The enzymatic degradation of dentin matrix turns collagen molecules into small peptide fragments. The latter leaches out from insoluble collagen matrix, resulting in decreased mass of collagen matrix and elasticity of bulk material. Hence, dry mass loss and hydroxyproline (HYP) release of dentin collagen after collagenase challenge were used in this study as indirect measurements of enzymatic degradation resistance. The sustainability of the elasticity of dentin collagen matrix was also evaluated.

The dentin beams of each group were treated with corresponding etchants (Table S1 in Supporting information), and the collagen degradation rate was shown in Fig. 1a. Dentin beams from control groups (i.e., 10% PhA, 10% PhA + DMSO) lost as high as 64.2%–91.8% of their original mass. There was no significant difference between the 10% PhA group and the 10% PhA + DMSO group (P > 0.05). Compared with the control groups, the mass loss of dentin treated by NDGA or PA-modified etchants was 24.9%–35.4%, which is significantly lower than that of control groups (P < 0.001). It means that the NDGA or PA groups have less collagen matrix degraded. Meanwhile, the concentration of PA or NDGA did not affect the dry mass loss of collagen matrix (P > 0.05).

Figure 1

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Fig. 1. (a) Loss of dry mass (percentage of original weight) from demineralized dentin matrix after treatment with different etchants. (b) HYP release (μg per mg of dentin) from demineralized dentin matrix after treatment with various etchants and challenged by collagenase for 48 h. Groups labeled with the same upper case letters are not statistically significant (P > 0.05).

We used release of HYP as a complementary indicator to estimate the percent dissolution of collagen peptide from collagen matrix after enzymatic degradation. This method was based on the assumption that 90% of the dry mass of the demineralized dentin beams consisted of type Ⅰ collagen [31] and that the dentin collagen contained 9.6% HYP [32]. Fig. 1b showed the release of HYP after 48 h of collagenase degradation in each group. The amount of HYP released from NDGA or PA-modified etchant was one order of magnitude lower than that of the control groups (P < 0.01). For the NDGA-modified etchant, the concentration did not influence the HYP release of collagen matrix (P > 0.05). However, PA-modified etchant reduced the HYP release in a concentration manner (P < 0.05). DMSO reduced HYP release in the control groups (P < 0.01). Both dry mass loss and HYP release results reveal that dentin beams demineralized by NDGA or PA-modified etchant have lower dissolution of collagen from demineralized dentin matrix than the control groups.

The modulus of dentin collagen with various intrafibrillar apatites ranges from 0.2 GPa to 2 GPa, whereas complete demineralization reduces its modulus from 0.008 GPa to 0.02 GPa [33]. The initial elastic modulus of each sample after corresponding etching treatment was shown in Fig. 2a. Compared with the control groups, the elastic modulus of dentin beams increased to some extent after the treatment of NDGA or PA-modified etchant (P < 0.05), which implies that crosslinking of collagen matrix did occur during the demineralization process. The loss of elasticity was depicted in Fig. 2b. The dentin matrix demineralized by NDGA or PA-modidied etchant showed sustained bulk mechanical property after biodegradation.

Figure 2

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Fig. 2. (a) The apparent modulus of elasticity of demineralized dentin beams treated with various etchants. (b) The apparent loss of elasticity of demineralized dentin beams treated with various etchants. Groups labeled with the same upper case letters are not statistically significant (P > 0.05).

In this study, the improved enzymatic degradation resistance and sustained mechanical properties of dentin collagen have demonstrated the collagen-stabilizing capability of bio-modified etchants. PA is a class of flavonoid oligomers composed of catechin/epicatechin building units. Due to the abundance of aromatic rings and phenolic hydroxyl groups in catechin/epicatechin units, PA is capable of stabilizing collagen matrix via the formation of multiple hydrogen bonds, hydrophobic interactions, as well as covalent-like bonds with collagen polypeptides [34]. NDGA could stabilize collagen by forming bridge-type hydrogen bonds between its phenolic hydroxyl and amide carbonyl groups of collagen, hydrophobic interaction (i.e., π-stacking) between aromatic rings, and covalent-like interactions, as did PA [35]. During crosslinking process, catechol moieties of NDGA would auto-oxidize into highly active o-quinone groups. NDGA may form bisquinone bond between two adjacent o-quinone groups, resulting in NDGA polymer matrix in which dentin collagen fibrils are embedded [25].

DMSO is polar aprotic solvent dissolving both polar and non-polar compounds. It is the best currently known penetration enhancer for medical purpose [36]. The tissue penetration capacity and excellent solvent properties make DMSO an attractive co-solvent for collagen crosslinker (i.e., PA, NDGA) in bio-modified etchants. In addition, previous studies have demonstrated that DMSO pretreatment of etched dentin could inhibit interfacial nanoleakage of resin-dentin bonds [37] and thus improve the bonding performance over time [38]. Indeed, in current study, adding DMSO into PhA has inhibited collagen degradation and improved the modulus of elasticity of dentin beams.

As the collagen-stabilizing capability of bio-modified etchants were validated by using dentin beam as macro-model of hybrid layer of resin-dentin bonding, we further characterized the etching effect of bio-modified etchants on flat coronal dentin, which simulated the clinical situation. As shown in Fig. 3, the etching depth in 10% PhA group was about 5 μm, while the 1% NDGA-modified etchant and 1% PA composite etchant etched dentin with about 2–4 μm, which, to some extent, was similar to the etching property of mild-acidic self-etching adhesives. The EDX spectra were taken from the demineralized region. It was shown that 10% PhA, with or without DMSO depleted both calcium and phosphate elements from dentin, whist dentin etched by bio-modified etchant displayed the presence of remnant calcium. It is well known that phenolic hydroxyl groups from PA or NDGA can interact with metal ions (i.e., calcium) via metal-coordination chemistry [39]. It is reasonable to speculate that the free calcium ions generated during the demineralization process of dentin might have participated in the crosslinking reaction of dentin collagen. This would further benefit the outcome of dentin bonding, as the calcium persisting in the collagen matrix may promote remineralization of denuded collagen fibrils that were not penetrated by adhesive resin [40].

Figure 3

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Fig. 3. Representative SEM (a-d) and EDX (e-h) images of HMDS-dehydrated demineralized dentin after dentin etching. (a, e) 10% PhA, (b, f) 10% PhA + DMSO, (c, g) 1% NDGA, (d, h) 1% PA. Scale bar: 2 μm.

In current study, dentin collagen matrix were demineralized and crosslinked simultaneously by bio-modified etchant. Compared to conventional methods, etch-and-crosslink technique has following merits: 1) It simplifies clinical complexity by combining etching and crosslinking steps; 2) It eliminates the possibility of compromised adhesive monomer conversion by unreacted crosslinker; 3) It enhances the biostability of dentin collagen and resin-dentin bonds over time. Bio-modified etchant with low concentration (0.5% or 1%) of NDGA can significantly improve the enzymatic degradation resistance and the stability of the mechanical properties of dentin collagen matrix. Our study validates the concept of etch-and-crosslink in dentin bonding. This simplified technique holds the potential to achieve durable resin-dentin bonding.

This work was supported by the Projects of the National Natural Science Foundation of China (No. 81571815 (Y. Liu), No. 81401525 (S.Q. Gong)), the Beijing Municipal Natural Science Foundation (No. 7152156 (Y. Liu)) and Beijing New-star Plan of Science and Technology (No. Z171100001117018 (Y. Liu)).

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.08.036.