On the stiffness of demineralized dentin matrices

Abstract

Resin bonding to dentin requires the use of self-etching primers or acid etching to decalcify the surface and expose a layer of collagen fibrils of the dentin matrix. Acid-etching reduces the stiffness of demineralized dentin from approximately 19 GPa-1 MPa, requiring that it floats in water to prevent it from collapsing during bonding procedures. Several publications show that crosslinking agents like gluteraladehyde, carbodiimide or grape seed extract can stiffen collagen and improve resin-dentin bond strength.

Objective: The objective was to assess a new approach for evaluating the changes in stiffness of decalcified dentin by polar solvents and a collagen cross-linker.

Methods: Fully demineralized dentin beams and sections of etched coronal dentin were subjected to indentation loading using a cylindrical flat indenter in water, and after treatment with ethanol or ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The stiffness was measured as a function of strain and as a function of loading rate from 1 to 50 μm/s.

Results: At a strain of 0.25% the elastic modulus of the fully demineralized dentin was approximately 0.20 MPa. It increased to over 0.90 MPa at strains of 1%. Exposure to ethanol caused an increase in elastic modulus of up to four times. Increasing the loading rate from 1 to 50 μm/s caused an increase in the apparent modulus of up to three times in both water and ethanol. EDC treatment caused increases in the stiffness in fully demineralized samples and in acid-etched demineralized dentin surfaces in situ.

Significance: Changes in the mechanical behavior of demineralized collagen matrices can be measured effectively under hydration via indentation with cylindrical flat indenters. This approach can be used for quantifying the effects of bonding treatments on the properties of decalcified dentin after acid etching, as well as to follow the loss of stiffness over time due to enzymatic degradation.

Keywords: Collagen; Crosslinking; Dentin bonding agents; Durability; EDC; Endogenous proteinases; Stiffness.

Figure 1

Schematic diagrams describing indentation of the dentin substrates using a cylindrical indenter with flat face. a) indentation of the fully demineralized dentin specimens; b) indentation of the acid-etched mineralized dentin specimens. The variable P, a, and h represent the indentation load, indenter radius and thickness of the demineralized substrate, respectively. The variable t in (b) represents the thickness of the etched dentin layer.

Figure 2

Load-displacement responses for indentation loading of the completely demineralized dentin samples in water. a) a single specimens with stiffness measurements at 0.25% and 1.0% strains; b) distribution of the stiffness responses obtained from the samples in water.

Figure 3

Influence of exposure to ethanol on the apparent stiffness of the demineralized dentin collagen matrix. The samples were initially evaluated in water. Then they were placed in a bath of 100% ethanol for 30 seconds and evaluated in ethanol. Thereafter, the samples were removed from ethanol, rehydrated in water for 15 minutes and evaluated again in water. a) comparison of the elastic modulus at 0.25% strain; b) comparison of the elastic modulus at 1% strain. The boxes for each condition represent the average and standard deviation. The error bars encompass the total range in responses and boxes with different letters indicate significant differences (p≤0.05).

Figure 4

Influence of loading rate on the elastic modulus of the demineralized dentin. Indentation tests were performed at loading rates of 1, 10 and 50 µm/sec. a) at 0.25% strain in water; b) at 0.25% strain in ethanol, c) at 1% strain in water, d) at 1% strain in ethanol. Note the difference in scales used for presenting the data in (a) – (d). Boxes with different letters indicate significant differences (p≤0.05).

Figure 4

Influence of loading rate on the elastic modulus of the demineralized dentin. Indentation tests were performed at loading rates of 1, 10 and 50 µm/sec. a) at 0.25% strain in water; b) at 0.25% strain in ethanol, c) at 1% strain in water, d) at 1% strain in ethanol. Note the difference in scales used for presenting the data in (a) – (d). Boxes with different letters indicate significant differences (p≤0.05).

Figure 5

Changes in stiffness of the demineralized collagen with EDC treatment for 1 minute. A comparison is made between the water and EDC responses at indentation strains of 0.25% and 1% (Figure 2(a)). Boxes with different letters indicate significant differences (p≤0.05).

Figure 6

The apparent stiffness of the etched dentin layer measured in situ under cylindrical indentation. a) comparison of the load-displacement responses for a sample evaluated in water and then after EDC treatment; b) elastic modulus measurements at 10% strain. According to a one-way ANOVA, the difference in elastic modulus between the two conditions is approaching significance (p=0.08).