Microhardness and microstructural properties of a mixture of hydroxyapatite and β-tricalcium phosphate

ABSTRACT

Calcium phosphate ceramics have been studied as promising materials for biomaterial applications owing to their excellent biocompatibility and osteoconductivity. Herein, we investigated the influence of different mass ratios of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) in a calcium phosphate mixture (CPM) on the microhardness and microstructural properties of a series of xHA-(100-x)β-TCP (x = 0, 30, 50, 70, 90, and 100) mixture samples. The chemical compositions and structural properties of the CPM samples were characterized using X-ray diffraction, Fourier-transform infrared spectroscopy, field emission scanning electron microscopy, and energy-dispersive X-ray spectroscopy. The porosity of the HA/β-TCP composites decreased with increasing β-TCP content and reached a minimum porosity when the mass ratio of HA/β-TCP is 70/30, and then increased with increasing β-TCP content again. The surface microhardness of the CPM composites was measured and found to be inversely proportional to their porosities. Therefore, the CPM of HA/β-TCP with a mass ratio of 70/30 exhibited a maximum surface microhardness of 86.02 MPa.

KEYWORDS:

• Hydroxyapatite
• β-tricalcium phosphate
• solid state reaction

1. Introduction

Life expectancy for humans has been increasing, and bone tissue engineering using biomaterials for bone tissue defects is gaining traction. Currently, calcium phosphates have attracted attention for bone grafting applications owing to their high osteoconductivity [1–3]. One of the most important advantages of calcium phosphates is their similarity to the mineral phase of the human bone. Calcium phosphates such as hydroxyapatite (HA), α-tricalcium phosphate (α-TCP), biphasic calcium phosphate (BCP), and β-tricalcium phosphate (β-TCP) are widely used as bone-grafting materials for bone regeneration in dental and orthopedic applications. A mixture of HA and β-TCP has been widely used in dental applications, like a mixture of calcium silicates (dicalcium silicate and tricalcium silicate) [4].

Particularly, porous HA has been studied because it aids in generating new bones that can be employed for tissue implantation [5]. Further, it has been used to control the volume of pores [6,7]. However, as an inert implant, HA possesses low biodegradability and poor mechanical stability, and is a high-temperature phase of calcium phosphate, whereas β-TCP is a low-cost resorbable bioceramic [8,9]. Therefore, many studies on enhancing the properties of biphasic calcium phosphate (composite of HA and β-TCP) have focused on different contents of β-TCP based on the HA matrix to increase mechanical stability. Optimum HA/β-TCP mass ratios of 70/30 and 60/40 have often been reported [10–12].

The effects of HA and β-TCP biphasic porous ceramics on the phase content and pore size for biomedical applications have been studied previously [13–15]. However, detailed and systematic research on the effect of the ratio of HA and β-TCP on the calcium phosphate mixture (CPM) in controlling its microstructural and mechanical properties has not been conducted. In ref [10], the microstructural and mechanical properties of various compositions of calcium phosphate scaffolds were investigated. However, the investigated phase compositions include HA, β-TCP, and α-TCP, which are different from our compositions that include only HA and β-TCP. Ref [11] and [12] reported the effect of the ratio of HA and β-TCP on the biocompatibility such as osseointegration, cell proliferation, and biodegradation, not on the mechanical properties. This study compares the microhardness and microstructures of HA/β-TCP mixture samples with different mass ratios. We prepared xHA-(100-x)β-TCP mixtures (x = 0, 30, 50, 70, 90, and 100); hence, this study focused on the influence of the HA content on the microhardness and microstructures of HA/β-TCP mixtures.

2. Materials and methods

2.1. Synthesis of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP)

HA and β-TCP were prepared by the solid-state reaction of calcium carbonate with ammonium phosphate monobasic. The Ca/P molar ratios of 1.67 and 1.5, which are the desired Ca/P ratios for synthesizing HA and β-TCP, respectively, were maintained. The starting materials, calcium carbonate (CaCO₃, 99%, Sigma-Aldrich) and ammonium phosphate monobasic (NH₄H₂PO₄, 99%, Sigma-Aldrich), were weighed and mixed by ball milling using alumina balls with the ball-to-powder weight ratio of 3.86 in acetone solvent for 24 h. Then, the powders were dried at 70°C for 24 h. The dried powders were sintered at 1200°C for 2 h and at 900°C for 10 h for HA and β-TCP, respectively. The synthesized HA/β-TCP lumps were crushed manually using an agate mortar and pestle, and sieved through a 400 mesh sieve. The mass change and thermal behavior of the raw materials were determined by thermogravimetry (TG) and differential thermal analysis (DTA) using a thermogravimetric differential thermal analyzer (TG-DTA, STA409PC2, Netzsch, Germany) at a heating rate of 10°C/min in air from 30°C to 1000°C. The crystalline structure was characterized using X-ray diffraction (XRD, DMAX-2500, Rigaku, Japan) at 40 kV and 100 mA in the 2θ range of 20–60° at a scan speed of 1°/min using Cu K_α1 radiation.

2.2. Fabrication of calcium phosphate mixtures (CPM) and physicochemical characterization

Six types of CPM (a mixture of HA and β-TCP) were prepared by changing the mass ratio of HA/β-TCP (denoted as HA, 90 HA, 70 HA, 50 HA, 30 HA, and TCP). The structure of the CPM was analyzed using Fourier transform infrared (FT-IR) spectroscopy (VERTEX 80 V, Bruker, Germany) with test wavelengths between 500 and 4000 cm⁻¹. The HA/TCP mixtures were uniaxially pressed in a 10-mm-diameter mold at 10 MPa. The CPM green bodies were sintered in furnace at 800°C for 2 h. The surface microstructures of the HA and β-TCP were examined by field emission scanning electron microscopy (FE-SEM, SU8010, Hitachi, Japan). The Ca/P ratios of the CPM were determined using energy-dispersive X-ray spectroscopy (EDX) analysis. The pore size and porosity (density of pores) were quantified by selecting five images from each specimen, and measuring the parameters using an image analysis software (Image-Pro Plus, Version 6.0, Media Cybernetics Inc., USA). The Vickers hardness was determined using a microhardness tester (MVK-H1, Shimadzu Co., Japan) under a load of 1000 gf for a loading time of 15 s. This procedure was repeated seven times, and the average hardness value was recorded for each sample.

3. Results and discussion

To determine the calcination temperature of calcium carbonate and ammonium phosphate monobasic, TG/DTA curves of the raw materials were obtained and are shown in Figure 1. The exothermic peak at approximately 800°C and mass loss in the TG curve of CaCO₃ indicate the decomposition of calcium carbonate (Figure 1(a)). The exothermic peak observed at 208°C in the DTA curve of NH₄H₂PO₄ is attributed to the decomposition temperature of the crystal. The mass loss in the TG curve of NH₄H₂PO₄ until a temperature of approximately 600°C confirms the dissociation of the substance and evaporation of NH₃ and H₂O. Subsequently, P₂O₅ evaporates above 660°C. The TG/DTA results show that much of the raw materials (CaCO₃ and NH₄H₂PO₄) are decomposed above approximately 800°C. Therefore, the appropriate calcination temperature for synthesizing HA and β-TCP is above 800°C.

Figure 1. Thermogravimetric and differential thermal analyses (TG/DTA) of (a) CaCO₃ and (b) NH₄H₂PO_4.

The XRD patterns of the products prepared by the calcination of calcium carbonate and ammonium phosphate monobasic powder at different temperatures are shown in Figure 2(a). The XRD pattern of the sample calcined at 1000°C exhibits two distinct phases: HA (Ca₅(PO₄)₃(OH), hexagonal, JCPDS file No. 00-009-0432) and β-TCP (Ca₃(PO₄)₂, rhombohedral, JCPDS file No. 00-009-0169). On the other hand, the crystalline structure of the sample calcined at 1200°C was identified as the pure HA phase, corresponding to JCPDS file No. 00-009-0432. Figure 2(b) shows the XRD patterns of the β-TCP samples calcined at 700°C, 800°C, and 900°C. The XRD patterns of samples calcined at 800°C and 900°C were indexed to the pure β-TCP phase corresponding to JCPDS file No. 00-009-0169, whereas Ca₂P₂O₇ and β-TCP phases were detected together for the sample calcined at 700°C. Table 1 shows the lattice parameters a and c of HA and β-TCP calculated from XRD data. The unit cell volume (V) and density (D_x) of HA and β-TCP were also calculated based on the calculated lattice parameters. The calculated D_x of HA and β-TCP were 3.11 and 3.07, respectively. The average crystallite sizes of HA and β-TCP calculated from the XRD data were 64.64 nm and 50.56 nm, respectively.

Figure 2. XRD patterns of (a) HA and (b) β-TCP synthesized at different temperatures by the reaction of CaCO₃ and NH₄H₂PO_4.

Table 1. Calculated lattice parameters, unit cell volumes, and densities of HA and β-TCP.

Sample	Lattice parameters (Å)		Unit cell volume V (Å^3)	Density Dx (g/cm^−3)
	a	c
HA	9.41	6.87	526.83	3.11
β-TCP	10.43	37.39	3522.53	3.07

The morphologies and dimensions of the initial HA and β-TCP powders are shown in Figure 3. Figures 3(a-d) present typical FE-SEM high-magnification and low-magnification images of the synthesized HA and β-TCP powders after calcination. HA comprises irregularly shaped agglomerated particles and the surface is heterogeneous, with some agglomerations on it. The observed particle sizes of β-TCP powders were approximately 3–5 μm.

Figure 3. FE-SEM morphology of the (a,b) HA and (c,d) β-TCP powders synthesized at 1200 and 800°C, respectively.

The XRD analysis of CPM with different HA/β-TCP ratios (HA, 90 HA, 70 HA, 50 HA, 30 HA, and TCP) is shown in Figure 4(a). The diffraction peaks of HA gradually decreased with increasing β-TCP content. Figure 4(b) shows the FT-IR spectra of all the CPM samples. The weak band centered at 3572 cm⁻¹ is attributed to the stretching mode of the hydroxyl group corresponding to that of hydroxyapatite [16] and decreased gradually with increasing β-TCP content. A small band at approximately 950 cm⁻¹ is assigned to v1, which is a non-degenerate P-O symmetric stretching mode. The band at 1039 cm⁻¹ is attributed to the v3 symmetric stretching mode. The band with the strongest intensity, which occurs in the range of 1209–946 cm⁻¹, belongs to the stretching modes of the PO^3-₄ group. For the β-TCP sample, the bands that appear at 605 and 551 cm⁻¹ correspond to the v4 bending mode. The results confirm the presence of HA and β-TCP mixtures.

Figure 4. (a) XRD patterns and (b) FT-IR spectra for different ratios of HA/β-TCP mixture (CPM).

The EDX spectra of HA and β-TCP are shown in Figure 5(a-b), respectively. The Ca/P ratios were calculated from EDX spectra for CPM with different HA/β-TCP ratios (HA, 90 HA, 70 HA, 50 HA, 30 HA, and TCP) as shown in Table 2. The Ca/P ratio decreases with increasing β-TCP content, confirming the mixing ratio of CPM. However, the Ca/P ratios of the samples that are slightly higher than stoichiometric Ca/P ratios suggest the presence of a small amount of CaO in the samples (~1 wt.%). The amount of CaO in the samples was very small; thus, it was not detected in XRD spectra. CaO can originate from thermal decomposition of CaCO₃ during solid-state reaction [17,18].

Figure 5. EDX spectra of (a) HA and (b) β-TCP powders synthesized at 1200 and 800°C, respectively.

Table 2. Results of calculation of Ca/P ratio based on EDX analysis.

Sample	StoichiometricCa/P ratio	EDXCa/P ratio
HA	1.67	1.71
90 HA	1.65	1.68
70 HA	1.62	1.63
50 HA	1.58	1.60
30 HA	1.55	1.58
TCP	1.5	1.54

Cross-sections of the HA/β-TCP composites sintered at 800°C were observed by FE-SEM, as shown in Figure 6. The pore sizes ranged from 0.4 to 0.5 μm, and partially isolated HA/β-TCP particles were observed. Figure 7(a) shows the porosities quantitatively calculated using an image analysis technique. The porosities of pure HA and β-TCP exhibited a similar value and were reduced in the mixture samples (90 HA, 70 HA, 50 HA, and 30 HA). The porosity decreased with β-TCP content until it reached a minimum value of 6.39% for 70 HA and then increased again. The porosity of a binary mixture of irregularly shaped particles is closely related to the equivalent packing diameter, d_p [19]:

Figure 6. FE-SEM cross-sectional images of (a) HA, (b) 90 HA, (c) 70 HA, (d) 50 HA, (e) 30 HA, and (f) TCP sintered at 800°C for 2 h.

Figure 7. (a) Porosity and (b) pore size distribution depending on the surface microhardness of xHA-(100-x)β-TCP (x = 0, 30, 50, 70, 90, and 100) CPM.

(1) $d_{\mathrm{p}}=\left(3.1781-3.6821\frac{1}{\mathrm{\Psi }}+1.5040\frac{1}{{\mathrm{\Psi }}^2}\right)d_{\mathrm{v}},$(1)

where d_v and Ψ are the equivalent volume diameter and sphericity, respectively. d_p can be defined as the diameter of a sphere that has original specific volume of a particle. HA and β-TCP have different morphologies, which can be influenced by various factors such as different crystal structures. As shown in Table 1, HA and β-TCP have different lattice parameters, unit cell volumes, and densities, which can cause differences in radii and sphericity that are the required input parameters for equation (1)(1) $d_{\mathrm{p}}=\left(3.1781-3.6821\frac{1}{\mathrm{\Psi }}+1.5040\frac{1}{{\mathrm{\Psi }}^2}\right)d_{\mathrm{v}},$(1) . The mixture of two particles with different morphologies can be considered as a model for binary mixtures of two different sizes of spheres with diameters of d_p. In such a model, the relationship between porosity and volume fraction exhibits a “V-shape” trend, in which the mixture has its minimum porosity (maximum packing density) at a critical volume fraction [20,21]. Therefore, 70 HA is the nearest to the critical volume fraction for the minimum porosity. The pore size distributions of the different HA contents are shown in Figure 7(b). The narrowest pore size distribution was observed for 70 HA, which is consistent with a previous report [22].

The mean surface hardness versus the HA/β-TCP ratio is shown in Figure 8. The mean surface hardness of HA and β-TCP are 19.19 MPa and 51.00 MPa, respectively. The measured surface hardness for HA was very low compared to the typical surface hardness for HA [23,24], which is attributable to relatively high porosity (6–15%) of the samples. Generally, surface hardness decreases with an increase in porosity of materials primarily due to the pores within the material collapsing under load [25–27]. The mixture samples (90 HA, 70 HA, 50 HA, and 30 HA) exhibited higher surface hardness than either HA or TCP. Compared to HA, the low content of β-TCP in HA enhanced its mechanical properties. The surface hardness increases until it reaches a maximum hardness value of 86.02 MPa (when the mass ratio of HA/β-TCP is 70/30), and then decreases with increasing β-TCP content. This result is consistent with the porosity trend, considering the inverse proportion to hardness of porosity. In addition, it is in accordance with the previous research reported by Shuai et al., in which a scaffold made of a HA/β-TCP mixture with a mass ratio of 70/30 exhibited optimum fracture toughness and compressive strength [28]. They elucidated their result by using the ceramic–ceramic composite approach [28–30]. When the β-TCP content in HA is low, it acts as a reinforcement dispersed in the HA matrix to improve the surface hardness. This approach can also be taken to explain that an increase in the hardness for 70 HA compared to HA was significantly large (approximately 4.5 times).

Figure 8. Surface microhardness of xHA-(100-x)β-TCP (x = 0, 30, 50, 70, 90, and 100) CPM sintered at 800°C for 2 h.

4. Conclusion

Hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) powders were synthesized by the solid-state reaction of NH₄H₂PO₄ with CaCO₃ in Ca/P molar ratios of 1.67 and 1.5, respectively. The optimum heat treatment conditions were calcination for 2 h at 1200°C and 10 h at 900°C for HA and β-TCP, respectively. The chemical compositions and structural properties of the CPM samples were investigated by XRD, FT-IR spectroscopy, FE-SEM, and EDX analysis. The porosity of the HA/β-TCP composites decreased with β-TCP content and reached a minimum porosity when the mass ratio of HA/β-TCP is 70/30, and then increased with β-TCP content again. The measured surface microhardness of the CPM composites followed the same trend in porosity. Therefore, the maximum surface microhardness was achieved for the HA/β-TCP mixture with a mass ratio of 70/30 (70 HA) sample. This study confirmed the possibility of controlling the mechanical and structural properties of the HA/β-TCP composites by adjusting the HA/β-TCP ratio. In the case of block-type bone grafting materials used for bone reconstruction, such as onlay and inlay grafting, the mechanical properties are significant because they are required to withstand heavy loads. Therefore, desirable mechanical properties of block-type bone grafting materials can be achieved by appropriate tuning of HA/β-TCP ratio.

Acknowledgments

This work was supported by the Technology development Program (S3319649) funded by the Ministry of SMEs and Startups (MSS, Korea).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data available on request from the authors.