Tuesday, October 3, 2023

Calcium phosphate ceramic as a model for enamel substitute material in dental applications – BDJ Open

Hydroxyapatite, tricalcium phosphate (TCP), and biphasic calcium phosphate (BCP) preparation

Hydroxyapatite and TCP powder synthesis

The starting materials in this study were CaCO3 (Fluka Chemie GmbH, Buchs, Switzerland) and CaHPO4 (Sigma Aldrich Chemie GmbH, München, Germany) due to chemical stability and well distribution of particle sizes [4]. All reagents were analytical reagent grade and were used as received. Deionized water (DI) was used in all processing steps. The starting materials CaCO3 and CaHPO4 with Ca/P molar ratios of 1.67 and 1.5 were used to produce pure hydroxyapatite and TCP powder following Eqs. (1) and (2) via a mechanochemical method [6].

$$\left( {{{{{{{{\mathrm{for}}}}}}}}\,{{{{{{{\mathrm{hydroxyapatite}}}}}}}}} \right)\,2{{{{{{{\mathrm{CaCO}}}}}}}}_3 + 3{{{{{{{\mathrm{CaHPO}}}}}}}}_4\; = > \;{{{{{{{\mathrm{Ca}}}}}}}}_{10}\left( {{{{{{{{\mathrm{PO}}}}}}}}_4} \right)_6\left( {{{{{{{{\mathrm{OH}}}}}}}}} \right)_2 + {{{{{{{\mathrm{H}}}}}}}}_2{{{{{{{\mathrm{O}}}}}}}} + 2{{{{{{{\mathrm{CO}}}}}}}}_2$$


$$\left( {{{{{{{{\mathrm{for}}}}}}}}\,{{{{{{{\mathrm{TCP}}}}}}}}} \right)\quad \quad {{{{{{{\mathrm{CaCO}}}}}}}}_3 + {{{{{{{\mathrm{CaHPO}}}}}}}}_4 = > {{{{{{{\mathrm{Ca}}}}}}}}_3\left( {{{{{{{{\mathrm{PO}}}}}}}}_4} \right)_2 + {{{{{{{\mathrm{H}}}}}}}}_2{{{{{{{\mathrm{O}}}}}}}} + 2{{{{{{{\mathrm{CO}}}}}}}}_2$$


CaCO3 and CaHPO4 were mixed and then ball milled using zirconia balls in DI and ethanol as a medium for 48 h for the synthesis of hydroxyapatite and TCP, respectively. The particles were dried in a hot air oven at 100 °C for 24 h. The as-synthesized hydroxyapatite and TCP powder particle size was approximately 4.5 µm measured by Zetasizer (Malvern Panalytical, Malvern, UK).

Dense sample preparation

The as-synthesized hydroxyapatite powder and the mixture of as-synthesized hydroxyapatite and as-synthesized TCP at a ratio of 70:30, so-called biphasic calcium phosphate (BCP) powder, were compacted under a uniaxial pressing of 10 MPa (HA10, BCP10) and 17 MPa (HA17, BCP17) for a dwell time of 60 s to consolidate the specimens into a bar with dimensions of 16 × 5 × 2 mm for the compressive strength test. For the Vickers microhardness test, the pellets (13 mm diameter and 3 mm thickness) were fabricated. The green compact was sintered by a two-step heat treatment in a normal atmosphere at 400 °C with a 100 °C per hour heating rate and 2 h holding time. Then, the temperature was increased to 1250 °C with the same heating rate of 100 °C per hour and 2 h holding time, followed by a furnace cooling to room temperature.

Human tooth preparation

The protocol was conducted in accordance with the Declaration of Helsinki and approved by the Human Research Ethical Committee of the Faculty of Dentistry, Chulalongkorn University (approval No. HREC-DCU 2022-075). Human premolars extracted according to patients’ treatment plans were collected. All teeth were inspected with a stereomicroscope (Stereo Microscope SZ61, Olympus, Tokyo, Japan) for cracks, decay, and restorations. The teeth were cleaned with a dental scaler and polished with fine pumice slurry using a low-speed handpiece before being stored in a 0.1% thymol solution at 37 °C for disinfection.

Compressive strength test

Only cusp tip areas in a coronal part of the tooth were used for the compressive strength test. The tooth was cut mesiodistally using a diamond saw blade (Isomet1000, Buehler, IL, USA) into ten enamel and dentin blocks with dimensions of 2 × 1 × 1 mm, which were confirmed by a digital caliper (CD-15AX, Mitutoyo, Kanagawa, Japan) before storage in 37 °C artificial saliva until use (Fig. 1).

Fig. 1: Tooth preparation diagram.

Preparation of enamel and dentin block for the compressive strength test.

The hydroxyapatite bars were cut into 10 small blocks of 2 × 1 × 1 mm. dimensions, which were confirmed by a digital caliper (CD-15AX, Mitutoyo, Kanagawa, Japan) (Fig. 2). A universal testing machine (LR10K, LLOYD Instruments, Bognor Regis, UK) with a 1000 N load cell and 0.5 mm/min crosshead speed was used. The compressive strength was calculated following Eq. (3), where F is the compressive strength value (MPa), P is the maximum load until material failure (N), and A is the cross-sectional area of the material resisting the load (mm2).

$${{{{{{{\mathrm{F = }}}}}}}}\frac{{{{{{{{\mathrm{P}}}}}}}}}{{{{{{{{\mathrm{A}}}}}}}}}$$


Fig. 2: Hydroxyapatite block preparation.
figure 2

Preparation of hydroxyapatite blocks for the compressive strength test.

Vickers microhardness test

For the microhardness test, 10 teeth were cut in a longitudinal section and mounted in an acrylic resin block. Wet silicon carbide abrasive papers with 600, 1000, 1200-grit, and alumina oxide polishing paste with 0.05 μm particle size were used for polishing to a smooth flat surface. The specimens were stored in 37 °C artificial saliva until use.

A Vickers microhardness tester (FM-810, FUTURE-TECH, Kanagawa, Japan) with a 50-g load and 10 s dwell time was applied on the surfaces of the specimens. Five indentations with 50 μm apart were located both occlusally and apically from a dentino-enamel junction of the tooth (Fig. 3A). For the hydroxyapatite groups, five indentations were applied (Fig. 3B). The length of each two diagonals of the square-shaped indentation was immediately measured by an optical microscopy with a 300 magnification and ±1 µm measurement error.

Fig. 3: Vickers microhardness test diagram.
figure 3

Vickers microhardness testing of (A) enamel–dentin and (B) hydroxyapatite specimens.

Specimen characterization using X-ray Diffraction (XRD) and scanning electron microscope (SEM)

The as-synthesized hydroxyapatite and as-synthesized TCP powders were analyzed phase formation by using X-ray Diffraction (XRD: PANalytical X’Pert Pro, PANalytical, Almelo, the Netherlands) with CuKα radiation (Kα = 1.5406 Å) operating at 30 mA and 40 kV. XRD was performed from 20 to 60° 2θ, at a step size of 0.02° 2θ and a scanning speed of 2.4° 2θ/min with a CuKα target. The spectra were analyzed using JADE software and JCPDS cards. The amorphous phase of as-synthesized hydroxyapatite and as-synthesized TCP powders confirmed the occurrence of hydroxyapatite (JCPDS No. 09-0432), and β-tricalcium phosphate (β-TCP) (JCPDS No. 09-0619) by XRD pattern.

In the same manner, both sintered hydroxyapatite and BCP in the dense form samples were also undergone an XRD (SmartLab®, Rigaku, Tokyo, Japan) with CuKα radiation (Kα = 1.5406 Å) operating at 30 mA and 40 kV from 20 to 60° 2θ, at a step size of 0.01° 2θ and at a scanning speed of 5° 2θ/min to confirm the formation of hydroxyapatite (JCPDS No. 09-0432) in HA10 and HA17 samples and the mixture of both hydroxyapatite (JCPDS No. 09-0432) and β-TCP (JCPDS No. 09-0619) in BCP10 and BCP17 samples. The proportions between hydroxyapatite and TCP in BCP samples were calculated by equations:

$${{{{{{{\mathrm{Hydroxyapatite}}}}}}}}\,\left( \% \right) = \frac{{{{{{{{{\mathrm{I}}}}}}}}_{{{{{{{{\mathrm{HA}}}}}}}}}}}{{{{{{{{{\mathrm{I}}}}}}}}_{{{{{{{{\mathrm{HA}}}}}}}}} + {{{{{{{\mathrm{I}}}}}}}}_{{{{{{{{\mathrm{TCP}}}}}}}}}}} \times 100$$


$${{{{{{{\mathrm{TCP}}}}}}}}\,\left( \% \right) = \frac{{{{{{{{{\mathrm{I}}}}}}}}_{{{{{{{{\mathrm{TCP}}}}}}}}}}}{{{{{{{{{\mathrm{I}}}}}}}}_{{{{{{{{\mathrm{HA}}}}}}}}} + {{{{{{{\mathrm{I}}}}}}}}_{{{{{{{{\mathrm{TCP}}}}}}}}}}} \times 100$$


The specimens were gold coated using a gold evaporation coating unit (JFC 1200, JEOL, Tokyo, Japan) before examining the morphology with a scanning electron microscope (Quanta250, FEI, USA). The SEM was operated at 20 kV and 10,000 magnification with a working distance optimized for imaging and large spot size.

Statistical analysis

SPSS version 28.0 software (IBM, Chicago, IL, USA) was used for all statistical tests. P-values less than 0.05 were considered statistically significant differences. A Shapiro–Wilk test was used to determine the normality of mean differences in compressive strength and microhardness values. Data were normally distributed (p > 0.05). Mean differences were analysed by one-way ANOVA and LSD post-hoc test.

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