Graphical abstract
Highlights
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A natural mineral porous media mimicking hypomineralized dentin is proposed.
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Dentin is deproteinized by heat treatment.
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The proposed porous media has a porosity of roughly 40%.
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The two pores sizes of the porous media are around 0.9 μm and 20 nm.
Abstract
Objectives
In order to evaluate the clinical impact of low viscosity resin infiltration in hypomineralized enamel, it is necessary to obtain a biomimetic porous substrate capable of mimicking enamel. The specifications for the biomimetic porous medium are defined using the literature data on hypomineralized enamel. Based on these specifications, we propose to use deproteinized dentin, the latter being deproteinized by heat treatment.
Methods and Results
Thermogravimetry analysis (TGA), field emission scanning electron microscopy (FESEM) observations, mercury intrusion porosimetry (MIP) tests and nanoindentation are performed on the deproteinized dentin tissue. Heat treatment is shown to be an effective and reproducible method for removing organic fluids and protein residues in dentin. Deproteinizing dentin also enables forming nanovoids by eliminating its organic matrix. The interconnected open nanoporosities (porosities of less than 100 nm) created at 600 °C are distributed between 14 nm and 32 nm and the total porosity is 39% (including 36% due to nanoporosities). At 800 °C, they are distributed between 60 nm and 100 nm and total porosity is 37% (including 33% arising from the nanoporosities). The hydroxyapatite crystal structure is transformed less at 600 °C, so this temperature should be preferred.
Significance
Besides providing new understanding of the dentin tissue itself, this study led to characterizing a porous medium made of natural apatite, and proposing and validating its use as a porous medium mimicking hypomineralized enamel. The next logical step of this study is the characterization of resin infiltration in this medium and its mechanical reinforcement.
1
Introduction
Enamel is the most mineralized tissue of the organism with a mineral component evaluated at 96% by weight and 87% by volume. However, the mineral component may be affected by certain pathologies. In the case of hypomineralization, the rate of mineralization decreases without loss of volume. In this case the enamel is called hypomineralized enamel. In hypomineralized enamel, the mineral part made up of hydroxyapatite crystals (Ca 10 (PO 4 ) 6 (OH) 2 ) is smaller than in sound enamel, reaching only 50–80% by volume. At the macroscopic scale, hypomineralized enamel becomes opaque, leaving unsightly white spots. At the microscopic scale, it presents interconnected porosity (20–200 nm) due to ultrastructure alteration (enlargement of the inter-prismatic sheath and localized acid dissolution of intra-prismatic crystals ). From the mechanical standpoint, hypomineralized enamel is weaker than sound enamel because mechanical properties like hardness and elastic modulus are strongly correlated with its degree of mineralization . Its hardness and elastic modulus are around 0.5 ± 0.3 GPa and 14.5 ± 7.5 GPa , respectively, versus 3–6 GPa and 70–115 GPa for sound enamel .
The objective of the resin infiltration technique, a new treatment fully compatible with the concept of minimally invasive dentistry , is to mask the white lesions of hypomineralized enamel. This treatment is based on the use of a photopolymerizable low viscosity resin with a refractive index close to that of sound enamel . Besides remaining a minimally invasive procedure , the treatment masks white opacity and reinforces the mechanical characteristics of hypomineralized enamel. The mineral network initially weakened is incorporated into a resin. It forms a semi-natural biocomposite with good mechanical properties, which is what makes it such an attractive biomimetic material. Regarding the new boom in therapeutic resin infiltration, an increasing number of infiltrants are expected on the market in the coming years, making it necessary to assess and compare them. Some of them have been studied separately from the lesion or by using a porous medium mimicking the lesion , that is to say the characteristics obtained (contact angle, surface tension, viscosity, hardening) do not take into account the medium invested. To bridge this gap, our aim is to define a reliable and reproducible model mimicking most of the characteristics of hypomineralized enamel (summarized in Table 1 ). This makes it necessary to perform infiltration and mechanical tests on pairs of resin/biomimetic porous medium.
| Characteristics | Expected range |
|---|---|
| Porosity | 20–40% |
| Pore size | 20–200 nm |
| Young’s modulus | 7–21 GPa |
| Other specifications | • Dimensions and homogeneity compatible with infiltration and mechanical tests. • Apatite shape close to the natural apatite shape found in enamel. |
The use of natural hypomineralized enamel seems ideal, but as hypomineralization is not an indicator for extraction, such samples are very difficult to gather in sufficient numbers for systematic testing. Therefore artificial hypomineralized enamel or models made of synthetic hydroxyapatite can be considered. However, these candidates must be dismissed because either they cannot provide a specimen with a calibrated volume (necessary for infiltration and mechanical tests) or the crystal form is not sufficiently close to that of natural crystal. As surprising as it may seem, deproteineized dentin could be a very interesting alternative in response to our specifications. First, dentin is formed by the same odontogenesis mechanisms as enamel. Enamel and dentin have the same hydroxyapatite chemistry. Secondly, the mineral density of dentin (50% by volume) is certainly lower than in sound enamel (87% by volume), but is still very close to that of hypomineralized enamel (60–70% by volume). Thirdly, the mechanical properties of dentin (hardness from 0.81 to 1.19 GPa and a Young’s modulus from 18 to 25 GPa) are close to those of hypomineralized enamel. Fourthly, the use of dentin samples offers the advantage of allowing the preparation of calibrated samples compatible with laboratory tests. However dentin presents an abundant collagen matrix (mainly composed of type I collagen fibers) strongly linked to the crystal lattice. To obtain a porous mineral medium from the dentinal substrate, we propose to deproteinize the dentin to remove the collagen fibers and only keep the mineral matrix. Given the complexity of the organic matrix, deproteinization by heat treatment seems more relevant and efficient than selective chemical deproteinization.
The objective of this study is to evaluate the morphological and mechanical consequences of dentin deproteinization by heat treatment to validate the use of deproteinized dentin as a substrate mimicking hypomineralized enamel.
2
Materials and methods
2.1
Sample preparation
Twenty non-carious human molars, extracted according to the protocols approved by the review board of the Dental Faculty of Paris-Descartes University, were used in this study. They were stored directly in 1% chloramine-T solution. The teeth were prepared using a flat wet grinding machine (Planopol-3, STRUERS) to retain only the coronal dentin. Enamel, cementum and radicular dentin were removed. Three teeth were kept for the control group (D0). Seventeen specimens were cut vertically into two equal parts to form two equivalent groups (D600 and D800). All the specimens were also acid etched with 37% phosphoric acid for 15 s and then rinsed to remove the smear layer formed during the preparation procedures.
A conventional gradient ethanol dehydration process was applied to the D0 specimens reserved for SEM observation, and for the specimens of groups D600 and D800. In groups D600 and D800, the samples were deproteinized by heat treatment (see next section). After the preparation procedure, all the specimens were stored in a desiccation room.
The specimens reserved for nanoindentation ( Fig. 1 ) were embedded before micron polishing. Specimens were rinsed in an ultrasound water bath (Transsonic 275, PROLABO) between each polishing step. Sound and hypomineralized enamel samples were also prepared in order to establish a comparison with the groups made from dentin. After embedding, the enamel samples and D0 group samples were stored in distilled water. Samples from groups D600 and D800 were again subjected to ethanol dehydration.
Sample preparation and the methodology are summarized in Fig. 1 .
2.2
Deproteinization by heat treatment (HT)
In groups D600 and D800, the samples were deproteinized according to the thermodynamic principle of phase transition. The deproteinization process was performed under air using the VITA INCERAMAT 3 oven. The samples were heated to 600 °C (D600) and 800 °C (D800), temperatures at which organic components are known to be eliminated while preserving mineral components. That is to say, the temperatures chosen were higher than 420 °C, the temperature at the organic matrix is degraded and lower than 850 °C , the temperature at which densification occurs due to the sintering behavior of hydroxyapatite grain.
2.3
Thermal analysis
Thermogravimetry and differential thermogravimetry analysis (TGA and DTGA) were carried out specifically on D0 group samples in an STA 449 F3 Jupiter (Netzsch). The sample weight of sound dentin (D0 group) used for thermogravimetry analysis was 17 mg. A heating rate of 5 °C/min was employed for the run and the temperature range was 30–1000 °C in a nitrogen and oxygen atmosphere of 80 ml/min and 60 ml/min, respectively.
2.4
Field emission scanning electron microscopy
Field emission scanning electron microscopy (FESEM) was used to compare the impact of the heat temperature (HT) on dentin qualitatively, especially in the crystal phase and for the porous microstructure, using an FEG LEO 1530 microscope (LEO Elektronen-mikroskopie GmbH) operated at low voltage (1–5 kV).
In each group, a specimen was randomly harvested and then fractured. The fractured samples were observed after being set on brass washers using a conductive coating.
2.5
Mercury intrusion porosimetry (MIP)
We used mercury porosimetry to quantify open porosity and assess the material pore size distribution. Mercury is a non-wetting liquid that will not naturally penetrate into the pore spaces of the sample. An incremental pressure was applied to the mercury so it entered the pores of the sample progressively. At each pressure step, the volume of mercury entering the sample was measured. The applied pressure P was linked to the pore diameter D via the surface tension of mercury γ and its contact angle θ using the Jurin–Laplace law:
D = − 4 γ cos θ P
A mercury porosimeter (Micromeritics Autopore IV, Micromeritics) was used with a small sample holder (Penetrometer 13-0609). The sample holder containing the dentin slices was weighed using an electronic balance with an accuracy of 0.1 mg (Sartorius TE214S, Labandco), then assembled on the porosimeter. The gas within the penetrometer was removed completely before the latter was filled with mercury under low pressure (approximately from 0.003 to 0.20 MPa, corresponding to the pore size between 420 and 6 μm). After removing the sample holder from the low pressure cell, the assembly (penetrometer, sample and mercury) was weighed. The different weight data were collected in order to use the penetrometer as a pycnometer. Then, the penetrometer (containing the sample immersed in mercury) was inserted in the high pressure cell. Pressures from 0.20 to 200 MPa (corresponding to pore sizes between 6 μm and 6 nm) were applied. The volume of mercury penetrating into the sample was recorded at each pressure step. The contact angle and surface tension of mercury were set beforehand for all the tests at 130° and 485 dyn/cm , respectively. To obtain reliable results, the manufacturer recommends a final mercury intrusion volume between 20 and 80% of the stem volume. To fulfill this condition, four samples, i.e. four half teeth, were used for each test. Four tests per group (D600 and D800) were performed: D600T1, D600T2, D600T3, D600T4 and D800T1, D800T2, D800T3, D800T4. As shown in Fig. 1 , groups D600 and D800 were obtained from the same teeth. The samples were fractured prior to testing in order to facilitate pore access.
2.6
Nanoindentation
The indentation experiments were performed using a nanoindenter (XP, MTS). The specimens were mounted on a support to ensure the exposure of a flat surface at right angles to the indenter. They were indented with a calibrated Berkovich indenter .
Samples of groups D0, D600 and D800 were mapped systematically with spacings of 100 μm between individual indentations, in order to scan a surface of 1 mm 2 , i.e. 100 acquisitions. The indenter tip was set to apply a load of 25 mN held for 5 s. The force–displacement curve of loading and unloading was used to calculate the mechanical properties (elastic modulus and hardness) of each indentation point.
The same procedure was also reproduced on sound and hypomineralized enamel samples.
2.7
Statistics
Porosity and mechanical property data were analyzed using the Kruskal–Wallis test, which is useful for the 2 by 2 comparison of batches with different variances. The significance level was fixed at p = 0.05.
2
Materials and methods
2.1
Sample preparation
Twenty non-carious human molars, extracted according to the protocols approved by the review board of the Dental Faculty of Paris-Descartes University, were used in this study. They were stored directly in 1% chloramine-T solution. The teeth were prepared using a flat wet grinding machine (Planopol-3, STRUERS) to retain only the coronal dentin. Enamel, cementum and radicular dentin were removed. Three teeth were kept for the control group (D0). Seventeen specimens were cut vertically into two equal parts to form two equivalent groups (D600 and D800). All the specimens were also acid etched with 37% phosphoric acid for 15 s and then rinsed to remove the smear layer formed during the preparation procedures.
A conventional gradient ethanol dehydration process was applied to the D0 specimens reserved for SEM observation, and for the specimens of groups D600 and D800. In groups D600 and D800, the samples were deproteinized by heat treatment (see next section). After the preparation procedure, all the specimens were stored in a desiccation room.
The specimens reserved for nanoindentation ( Fig. 1 ) were embedded before micron polishing. Specimens were rinsed in an ultrasound water bath (Transsonic 275, PROLABO) between each polishing step. Sound and hypomineralized enamel samples were also prepared in order to establish a comparison with the groups made from dentin. After embedding, the enamel samples and D0 group samples were stored in distilled water. Samples from groups D600 and D800 were again subjected to ethanol dehydration.
Sample preparation and the methodology are summarized in Fig. 1 .
2.2
Deproteinization by heat treatment (HT)
In groups D600 and D800, the samples were deproteinized according to the thermodynamic principle of phase transition. The deproteinization process was performed under air using the VITA INCERAMAT 3 oven. The samples were heated to 600 °C (D600) and 800 °C (D800), temperatures at which organic components are known to be eliminated while preserving mineral components. That is to say, the temperatures chosen were higher than 420 °C, the temperature at the organic matrix is degraded and lower than 850 °C , the temperature at which densification occurs due to the sintering behavior of hydroxyapatite grain.
2.3
Thermal analysis
Thermogravimetry and differential thermogravimetry analysis (TGA and DTGA) were carried out specifically on D0 group samples in an STA 449 F3 Jupiter (Netzsch). The sample weight of sound dentin (D0 group) used for thermogravimetry analysis was 17 mg. A heating rate of 5 °C/min was employed for the run and the temperature range was 30–1000 °C in a nitrogen and oxygen atmosphere of 80 ml/min and 60 ml/min, respectively.
2.4
Field emission scanning electron microscopy
Field emission scanning electron microscopy (FESEM) was used to compare the impact of the heat temperature (HT) on dentin qualitatively, especially in the crystal phase and for the porous microstructure, using an FEG LEO 1530 microscope (LEO Elektronen-mikroskopie GmbH) operated at low voltage (1–5 kV).
In each group, a specimen was randomly harvested and then fractured. The fractured samples were observed after being set on brass washers using a conductive coating.
2.5
Mercury intrusion porosimetry (MIP)
We used mercury porosimetry to quantify open porosity and assess the material pore size distribution. Mercury is a non-wetting liquid that will not naturally penetrate into the pore spaces of the sample. An incremental pressure was applied to the mercury so it entered the pores of the sample progressively. At each pressure step, the volume of mercury entering the sample was measured. The applied pressure P was linked to the pore diameter D via the surface tension of mercury γ and its contact angle θ using the Jurin–Laplace law:
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