Ceramics International 47 (2021) 30061–30070

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Ceramics International
journal homepage: www.elsevier.com/locate/ceramint

Effects of microwave processing parameters on the properties of

nanohydroxyapatite: Structural, spectroscopic, hardness, and
toxicity studies
Y.W. Sari *, A. Saputra, A. Bahtiar, N.A. Nuzulia
Department of Physics, Faculty of Mathematics and Natural Sciences, IPB University, Bogor, 16680, Indonesia

A R T I C L E I N F O A B S T R A C T

Keywords: This paper analyzed the effects of the processing parameters on the structural, spectroscopic, and hardness
Biomaterials properties and toxicity of nanohydroxyapatite (nanoHAP) materials, specifically in terms of low-power short-
Calcium phosphate duration microwave irradiation. NanoHAP compounds were synthesized from duck eggshells (a bio-based cal­
Eggshells
cium source) and phosphoric acid via a wet precipitation method and subsequent treatment by microwave
Hydroxyapatite
Microwave treatment
irradiation. Three different power inputs of 40, 200, and 400 W were used for the microwave tests, which were
conducted for durations of 35, 40, and 45 min. The results confirmed the benefits of microwave treatments in
terms of rapid heating, reduced reaction time, and high throughput. The existence of pure nanoHAP was
confirmed by an X-ray diffractogram, and the high energy emitted by the microwave irradiation combined with
an extended reaction time was proven to further transform eggshell calcium (CaO) into nanoHAP. The shift
toward narrower and smaller particles with increasing irradiation power and treatment duration was confirmed
via scanning electron microscopy, and an increase in hardness with extended irradiation was demonstrated. A
toxicity analysis conducted on human embryonic cells confirmed high cell viability after incubation with
nanoHAP. This indicates that nanoHAP obtained from microwave irradiation treatment has the potential to be
used as a biomaterial.

1. Introduction material science and nanotechnology have further enhanced its bio­
logical properties [21]. Nanosized and nanocrystalline HAP can imitate
Hydroxyapatite (HAP) is a calcium phosphate ceramic material from the dimensions of natural calcium phosphate present in bones and teeth
the apatite family. It is the most stable calcium phosphate ceramic [22], and it has been suggested that the microstructural properties of
compound in terms of the pH, temperature, and composition of the nanohydroxyapatite (nanoHAP) influence its biocompatible properties.
physiological fluid [1] and is expressed stoichiometrically as The biocompatibility of HAP nanoparticles was improved with the use of
Ca10(PO4)6(OH)2. The similarities between the chemical and structural a rough surface [23], and well-crystallized nanoHAP exhibited little
properties of HAP and those of bone and tooth minerals [2] have led to bioresorption activity [21]. Therefore, the application of fine-tuned
the widespread use of HAP as a bone substitute and as a bone replace­ nanoHAP as a potential biomaterial is proposed, and understanding
ment material in the field of biomedicine [3–7]. the effects of material sciences and nanotechnologies on the micro­
HAP is used extensively in biomedical applications as it minimizes structural properties of nanoHAP will be useful.
the likelihood of donor compatibility issues, immune rejection, and Nanotechnological advances have been beneficial for the develop­
pathogen transfer in reconstructive procedures involving autografts or ment of HAP [2], and microwave irradiation is considered a promising
allografts [8]. Under such conditions, HAP has been used as a bone filler technique for producing HAP nanoparticles [24]. The application of an
[9–11], a tissue engineering scaffold [12–15], an implant coating ON/OFF working cycle (3 min ON and 5 min OFF for a total of 19 min)
[16–18], and a drug delivery material [19,20]. during a 600-W microwave treatment resulted in irregular nanosized
HAP is an ideal potential biomaterial due to its good biocompati­ HAP powders that agglomerated to form microsized particles (0.3–5.0
bility, bioactivity, and osteoconductivity, and advances in the fields of μm) [25]. High-density HAP compacts have been produced by

* Corresponding author.
E-mail address: [email protected] (Y.W. Sari).

https://doi.org/10.1016/j.ceramint.2021.07.182
Received 12 February 2021; Received in revised form 25 June 2021; Accepted 18 July 2021
Available online 20 July 2021
0272-8842/© 2021 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Y.W. Sari et al. Ceramics International 47 (2021) 30061–30070

subjecting HAP powder to a 3-kW microwave-assisted solid-state sin­ 2.2. Microwave irradiation
tering procedure. High-temperature sintering at temperatures of
1000 ◦ C, 1100 ◦ C, and 1150 ◦ C produced HAP compacts with grain sizes NanoHAP obtained from microwave irradiation (Samples B, C, and
of 0.17, 0.5, and 1.16 μm. Here, an increased sintering temperature D) was prepared using a similar procedure to Sample A. As the nanoHAP
resulted in larger-sized grains [26]. would become a compact specimen after microwave irradiation, the
Several precursors have been utilized as starting materials for HAP. processes of decantation, separation, and drying were omitted for the
Phosphorous precursors are largely synthetic, such as H3PO4 and microwave-irradiated nanoHAP. In this case, after the addition of 0.6-M
(NH4)2HPO4. Common synthetic calcium precursors include Ca H3PO4 to the CaO solution, the semiliquid mixture was exposed directly
(NO3)2⋅4H2O, Ca(OH)2, Ca(NO3)2, and CaCl2, and typically, calcium to microwave irradiation. The investigation used a modified domestic
precursors are derived from bio-based (particularly shell-based) mate­ microwave (Sharp, R-230 R) to test three different levels of microwave
rials, including oyster shells [27,28], mussel shells [29,30], and chicken irradiation inputs (40, 200, and 400 W). The samples were subjected to
eggshells [31,32]. HAP particles derived from mussel shell, clam shell, microwave irradiation for durations of 35, 40, and 45 min, and all tests
and eggshell precursors exhibited similar properties, which indicates the were conducted twice. Table 1 presents the sample code for the corre­
possibility of using different types of calcium sources as precursors [33]. sponding experimental conditions.
HAP has been synthesized from various bio-based calcium precursors
via microwave irradiation. Precursors derived from green mussel shells 2.3. XRD analysis
underwent a 30-min 1.1-kW microwave treatment, and this resulted in
HAPs with a dense agglomerated structure and nanorod morphology An XRD analysis was performed on all samples using a Panalytical
with a 15–20-nm width and a 30–70-nm length [34]. Recently, the Empyrean XRD diffractometer with CuKα radiation at 40 kV and 30 mA.
possibility of using chicken eggshells as HAP precursors has been The samples were scanned from 20 ◦ C to 80 ◦ C at increments of 0.02 ◦ C/
investigated. HAP was synthesized under the influence of an organic min. The obtained XRD spectra were compared with the specifications in
modifier (ethylenediaminetetraacetic acid) at different pH levels and the Joint Committee on Powder Diffraction Standards (JCPDS-09-0432).
under varying microwave power intensities [35]. That research explored The lattice parameters of the nanoHAP samples were calculated using
the effect of microwave power on HAP yield as the basis for upscaling Cohen’s method, and the crystallite size at the (002) plane was esti­
HAP production from the lab to the pilot scale. The results confirmed mated from the broadening of the XRD peak, which was calculated using
that a 900-W lab-scale microwave treatment produced a yield similar to Scherrer’s equation as follows [38,39]:
that of a 2.2-kW pilot microwave treatment. The duration of the pilot
scale treatment was observed to be half that required for the pilot scale. t(hkl) =
0.9λ
,
This paper analyzed the effects of microwave irradiation processing β cos θ(hkl)
on the structural, spectroscopic, and hardness properties and toxicity of
nanoHAP materials, specifically in terms of low-power short-duration where t(hkl) is the crystallite size, λ is the wavelength of the mono­
microwave irradiation. The experiment used duck eggshells as a calcium chromatic X-ray beam (λCu = 0.154056 nm), β is the full width at half
source. The conversion of eggshells into HA is important not only from maximum (FWHM), and θ(hkl) is the peak diffraction angle that satisfies
the perspectives of material science and engineering but also from an Bragg’s law for the (hkl) Miller’s plane. This plane (002) was selected for
industrial viewpoint in which wastes can be minimized and recycled the crystallite analysis on account of its sharpness and isolation.
into advanced materials [36]. X-ray diffraction (XRD) and scanning In the nanoHAP powders, the degree of crystallinity, which indicates
electron microscopy were used for the structural investigations. The the fraction of the crystalline phase (CIXRD), was calculated using the
effect of the processing parameters on nanoHAP functional groups was following equation:
analyzed by Fourier-transform infrared spectroscopy (FTIR), and 0.24 3
microhardness properties were investigated using a Vickers micro­ CIXRD = ( ),
β002
hardness tester. A tetrazolium salt (MTT) colorimetric assay was used for
the toxicity analysis. where β002 represents the FWHM of the (002) reflections.

2. Materials and methods 2.4. Fourier-transform infrared spectroscopy

NanoHAP was synthesized using duck eggshells (as a bio-based cal­ FTIR spectroscopy was conducted using Bruker Tensor 37 with a
cium source) and phosphoric acid (H3PO4, Merck). The eggshells were wavenumber of 4000–400 cm− 1. Using the transmission technique,
obtained from a domestic market. A triple-power-level microwave oven samples were prepared as standard potassium bromide (KBr) pellets. A
(Sharp, R-230 R) provided the microwave source. Eggshell calcination further analysis of the splitting of the phosphate ion antisymmetric band
was performed in a Nabertherm B180 furnace. The process of converting at 750 to 500 cm− 1 was conducted to reveal the crystallinity index (CI)
eggshells into calcium (in the form of CaO) was conducted in accordance obtained by FTIR spectroscopy [40]. Two methods for obtaining the
with a previous study [37]. FTIR CI were investigated using the area and splitting height on the
phosphate ion antisymmetric band.
2.1. Preparation of hydroxyapatite The CI from the FTIR study obtained using the height method,
hereafter called CIheight-FTIR, was estimated by drawing a baseline from
A wet precipitation method was utilized to prepare nanoHAP com­ 750 to 500 cm− 1 and measuring the heights of the two in-plane
pounds using CaO (from eggshells) and H3PO4 (obtained from Merck) as
the starting materials. A suspension of stoichiometric CaO in 100 mL of
Table 1
distilled water underwent vigorous stirring at ambient temperature (±
Sample code for the corresponding experimental conditions.
25 ◦ C). A 0.6-M H3PO4 solution was added in a dropwise manner to the
Power (W) Time (min)
CaO solution at a steady rate of 5 mL/min. Then, the precipitate was
decanted at room temperature for 20 h before the solids and liquid were 0 35 40 45
separated with Whatman grade 41 ashless filter paper (Merck). The 0 A
precipitation (Sample A) was dried overnight at a temperature of 60 ◦ C. 40 B1 B2 B3
200 C1 C2 C3
400 D1 D2 D3

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phosphate symmetrical bending bands (υ4 ) (at 603 and 565 cm− 1) and at
595 cm− 1 (the signal was at 595 cm− 1, which was the lowest point be­
tween 603 and 565 cm− 1) (see Fig. 1) [41,42]. With Ax as the absorbance
at a given wavelength, the CIheight was calculated using the following
formula:
A565 + A603
CIheight− FTIR = .
A595
Additionally, the CI was estimated using the area method. As in the
height-based approach, the baseline was drawn from 750 to 500 cm− 1.
For the visual estimation of the area of the A2 band, which represents the
antisymmetric absorption of phosphate ions, further image processing
was conducted using Fiji ImageJ software [40]. Then, a line was drawn
to connect the two split peaks of the bands. The area of A1 was deter­
mined as the area between these two peaks (see Fig. 2). The CI obtained
using this method, hereafter called CIarea-FTIR, was calculated in accor­
dance with the following formula:
A1
CIarea− FTIR = .
A2
Fig. 2. Crystalline index measurement using the area method based on the
2.5. Morphology splitting fraction measurement at the phosphate ion antisymmetric bending
frequency in the calcium phosphate.

A morphological analysis was conducted using an FEI Quanta 650

scanning electron microscope (SEM) energy-dispersive X-ray diffrac­ P
tometer with an operating voltage of 20 kV. In all samples, particle size HV = 1.8544
d2
,
and distribution were obtained using Fiji ImageJ image processing
software. The particle sizes were obtained from 40 points of the SEM where P is the applied test load in N, d is the arithmetic average of the
images. two diagonal lengths of the resultant impression in mm, and 1.8544 is
the geometrical constant of the diamond pyramid [44].
2.6. Hardness test
2.7. Toxicity test
A hardness test was performed on samples A and D (D1, D2, and D3).
The nanoHAP powders were compacted into cylindricall pellets (diam­ The cytotoxic effects of the nanoHAP samples were evaluated using a
eter = 1 cm, thickness = 0.5 cm) at 50 MPa before the test. A Vickers yellow tetrazolium salt (MTT) assay, and the number of living cells (cell
hardness test was conducted using an LM 800AT Vickers microhardness viability) was determined according to the presence of a blue formazan
tester. A test load of 99 gf (1 N) was applied to five points on the created during a specific exposure period. The formazan resulted from
specimen’s surface for a fixed dwell time of 10 s prior to the discon­ the cleavage of the MTT, which represented the reaction of living cells
nection of the indenter (a Vickers elongated diamond pyramid indenter with functional mitochondria [45]. In this assay, HEK293T cells were
under a × 50 objective lens). The test was conducted in accordance with cultured in a growth medium containing Dulbecco’s modified Eagle’s
the procedures outlined in the ASTM E384 standards [43]. The Vickers medium (Gibco, USA), 10% fetal bovine serum (Gibco, USA), and 1%
hardness value (HV) was calculated using the following equation: antibiotic (penicillin–streptomycin, Invitrogen, USA) in a suitable
environment (37 ◦ C, 5% CO2, and 90% humidity). The cells were sub­
cultured and seeded at a density of 5000 cells per well into 96-well
tissue-culture plates. They were left to adhere for 18–20 h in the
growth medium. The medium was removed from the wells the following
day and was replaced with the nanoHAP samples adhered in the growth
medium. Then, the plates were incubated for 48 h at various concen­
trations. MTT (Sigma, USA) was added to each plate well (10 μL in 5
mg/mL in PBS). The plates were incubated for a further 4 h. Following
incubation, the medium was removed, and 1-N HCl in isopropanol (100
μL) (Merck, USA) was added. Finally, the absorbance of the plates at
562 nm was observed using a BioRad (USA) microplate reader. The
absorbance values were blanked against 1-N HCl in isopropanol, and the
absorbance of those cells exposed solely to the growing medium was
taken as 100% cell viability (negative control). The following formula
was used to calculate the survival rate percentage (cell viability) [46]:
Mean OD of treated cells − Mean OD of blank
Cell viability (%)= × 100%.
Mean OD of untreated cells − Mean OD of blank

3. Results and analysis

Fig. 1. Crystalline index calculation using the height method based on the The use of microwave treatment in nanoHAP synthesis has been
splitting fraction measurement at the phosphate ion antisymmetric bending investigated using various methods, including precipitation, reflux,
frequency in the calcium phosphate. metathesis, combustion, and hydrothermal, solvothermal, and

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ultrasonic means [47]. This study explored the effects of microwave

treatment on nanoHAP precipitation, specifically using eggshell-based
calcium precursors. Microwave treatments were found to offer multi­
ple benefits in terms of rapid heating, short reaction time, and high
throughput, e.g., a 1-h reaction time produced 9.78 g of nanoHAP
powder (the D3 sample), and precipitation followed by 24-h decantation
without microwave treatment yielded 9.11 g (the A sample). A signifi­
cant increase in powder mass was obtained (P < 0.005) when the mi­
crowave irradiation power was increased (Fig. 3).

3.1. X-ray analysis

The crystalline components in the nanoHAP powder were identified,

and the compound’s crystal structure was analyzed using XRD. The
recorded diffractogram presented in Fig. 4 confirms the presence of a
crystalline hexagonal nanoHAP in accordance with JCPDS 09–0432. The
sintering process is widely employed in nanoHAP synthesis, resulting in
highly crystalline HAP [48]. However, by omitting this step in this
study, we anticipated getting a clear view of the effect of microwave
irradiation time and power on the physical properties of the nanoHAP
without bias from the sintering process. Although this study omitted the
sintering process, the natural presence of highly crystalline nanoHAP
was indicated by sharp peaks at 2θ = 31.793◦ .
Figs. 4 and 5 show that the most intense nanoHAP Bragg reflection
for all samples was observed at the (211) plane in accordance with
JCPDS 09–0432 and another study [49]. In that research, a 45-min
period of high-powered microwave irradiation up to 1200 W produced
preferential crystal growth [49]. With a power input of 1200 W and a
treatment time of 15 min, a shift in the preferential crystal growth from
the [211] to the [300] directions was noted, and 45 min microwave
irradiation power and time were applied, respectively. However,
changes in preferential crystal growth were not observed in the present
study. Neither an increase in microwave irradiation energy (up to 400
W) nor an extended reaction time (up to an additional 45 min) altered
the preferential crystal growth, which was in the [211] directions for all
samples.
As the product of eggshell calcination [37] and as the result of its
incomplete transformation into nanoHAP, CaO could be present. How­
ever, the existence of an extraneous peak at 2θ = 37.346◦ (JPPDS
37–1497) confirmed that not all samples exhibited the presence of CaO.
This indicates that further transformation of calcium (in the form of
CaO) into nanoHAP was achieved by the high energy emitted by
Fig. 4. X-ray diffractogram of nanoHAP synthesized without precipitation (A)
and with microwave treatment for irradiation times of a) 35 and b) 45 min. The
irradiation power values for samples B, C, and D were 40, 200, and 400 W,
respectively.

microwave irradiation and a prolonged reaction time. Furthermore, the

peak correlates with the secondary phases or intermediate calcium
phosphate compounds, such as CaHPO4⋅2H2O or Ca8 (HPO4)2(­
PO4)4⋅5H2O, and these were not observed in the diffractogram, which
indicates that phase-pure HAP was produced by the reaction [50]. The
diffractograms of the samples treated with the highest microwave power
(400 W) for different treatment durations are given in Fig. 5.
The crystallographic parameters obtained from all samples and
JCPDS 09–0432 as reference, are listed in Table 2. The lattice parame­
ters of nanoHAP obtained with and without microwave irradiation had a
good accuracy with the reference. This good accuracy indicates the
purity of the nanoHAP obtained using both the methods.
Changes in the structural properties of the nanoHAP powders were
observed from the crystallite size calculations (Table 2). The grain size of
nanoHAP powder is a key structural parameter that influences most of
the material properties. Therefore, the crystallite size was calculated.
Fig. 3. Mass of nanohydroxyapatite (nanoHAP) synthesized without micro­ The width of the Bragg reflection can be used to estimate the grain size.
wave treatment (A) and with microwave treatments (B, C, and D). The small Assuming that the changes in the peak width are attributed to the size
letters on the chart indicate significant differences between samples (P < 0.05).

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Fig. 5. Diffractogram of samples irradiated at 400 W for different durations:

D1: 35 min, D2: 40 min, and D3: 45 min.

Table 2
Crystallographic parameters of nanoHAP powder and its crystallite size at (002).
Samples Lattice parameters Crystallite
size at (002)a
a=b accuracy c (Å) accuracy
(nm)
(Å) (%) (%)

Ref (JCPDS 9.418 6.884

09–0432)
A 9.432 ± 99.85 6.880 ± 99.94 16.72 ± 0.14a
0.002 0.002
B1 9.438 ± 99.79 6.882 ± 99.97 17.91 ± 0.17b
0.002 0.002
B2 9.440 ± 99.77 6.881 ± 99.96 17.47 ± 0.17c Fig. 6. Fourier-transform infrared spectra of nanoHAP synthesized without
0.002 0.002 precipitation (A) and with microwave treatment for irradiation times of a) 35
B3 9.443 ± 99.73 6.884 ± 100 17.4 ± 0.16c and b) 45 min. The irradiation power values for samples B, C, and D were 40,
0.002 0.002 200, and 400 W, respectively.
C1 9.434 ± 99.83 6.876 ± 99.88 18.00 ± 0.16b
0.002 0.002
C2 9.436 ± 99.81 6.878 ± 99.91 19.25 ± 0.19d
0.002 0.002
C3 9.447 ± 99.69 6.886 ± 99.97 19.57 ± 0.18d,
e
0.002 0.001
D1 9.433 ± 99.84 6.884 ± 100 20.95 ± 0.17f
0.002 0.001
D2 9.434 ± 99.83 6.886 ± 99.97 19.82 ± 0.18e
0.002 0.001
D3 9.431 ± 99.86 6.881 ± 99.96 19.89 ± 0.19e
0.002 0.001
a
Values with different superscript numbers indicate significant differences
between samples.

effect, the width of the reflection will decrease as the grain size increases
[1]. A one-way ANOVA test with a Duncan post-hoc test revealed that in
all samples, an increased crystallite size corresponded with the intensity
of the microwave irradiation power (P < 0.005). The microwave irra­
diation time impacted the crystallite size; however, we could not iden­
tify the trends in this study. To observe the trend, further study with
more time-variation could be performed.
Fig. 7. Fourier-transform spectra of nanoHAP synthesized with microwave
treatment at 400 W for treatment times of (D1) 35, (D2) 40, and (D3) 45 min.
3.2. Fourier-transform infrared analysis
functional groups of stoichiometric HAP [51]. The presence of atmo­
The FTIR spectra of nanoHAP synthesized in this study are repro­ spheric CO2 is indicated by the small vibrational peaks observed at
duced in Figs. 6 and 7. Those in the range of 400–4000 cm− 1 exhibited around 870 and 1420–1450 cm− 1 [52,53]. In addition, a broad band at
the characteristic bands of PO3−4 and OH that correspond to these
− 3500–2500 cm− 1 and a sharp peak at 1645 cm− 1 were observed, and

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these correspond to the OH− stretching and deformation vibrational Table 4

modes of the adsorbed and/or bound H2O on the surface of the HAP [52, Crystallinity index obtained from X-ray diffraction and Fourier-transform
54,55]. All infrared vibrations observed in this study are listed in infrared spectroscopy analyses.
Table 3. Sample CIXRD CIheight-FTIR CIarea-FTIR
CI data produced by FTIR spectroscopy were used to indicate the A 0.66 3.38 0.042
crystallinity (%) of calcium phosphate [40]. Table 4 displays the CI B1 0.67 4.02 0.034
obtained using the height method and by calculating the splitting factor B2 0.69 4.30 0.076
at the phosphate asymmetric bending bands (CIheight-FTIR), and it also B3 0.89 4.34 0.056
C1 0.62 4.04 0.068
shows the CI obtained using the area method (CIheight-FTIR) and from the
C2 0.76 4.93 0.116
XRD analysis, namely, CIFTIR and CIXRD, respectively. Both CI calcula­ C3 0.92 4.96 0.045
tions revealed that increased microwave power and extended treatment D1 0.84 4.95 0.049
time produced an increase in crystallinity. This finding is similar to the D2 0.88 5.12 0.029
results of a previous study in which the crystallinity of synthetic HAP D3 0.93 5.25 0.055

increased with higher heating temperature [41].

However, when the area method was used to calculate the CI, this synthesized sample (Sample B). No significant difference in particle size
trend was not observed. Not all of the CI methods by FTIR exhibited a was observed when compared with the sample synthesized without
similar pattern to the CI obtained from XRD, indicating that different microwave irradiation, i.e., without the introduction of additional en­
mechanisms are calculated from different analytical approaches. For ergy. An extended irradiation duration of up to 45 min had no significant
example, the CI obtained from XRD is related to the structural influence on nanoHAP particle size at this low temperature.
arrangement of the samples, and the index produced by FTIR is related There is a correlation between high microwave irradiation power
to the crystal bonds of the specimens [41]. Therefore, the CI data ob­ and high reaction temperature. Under conditions of high reaction tem­
tained from XRD are not comparable with FTIR-derived data. Further­ perature, the viscosity of reaction systems is decreased, and this accel­
more, as the result obtained in this analysis corresponds with that of the erates the collision rate of nuclei, which results in a smaller particle size
previous study, it is suggested that FTIR-derived CI obtained using [60]. Neither the application of the highest microwave irradiation
height methods could be valuable in the qualitative analysis of crystal­ power nor the duration of irradiation initiated the agglomeration of
linity (%) with respect to the noncrystalline phase. nanoHAP particles. During coalescence, the crystallites serve as nucle­
ation centers between which new bonds are formed [64]. Consequently,
the particle size increases, but this phenomenon was not observed in the
3.3. Scanning electron microscopy analysis
present study.
Fig. 10 shows the size distribution of the nanoHAP obtained in this
The SEM images of all of the samples obtained using the secondary
research. For the nanoHAP synthesized without microwave treatment,
electron mode are shown in Fig. 8, and a similar morphological trend is
particle sizes ranged between 80 and 150 nm. There was a shift in the
evident. All nanoHAP samples exhibited an unusual spherical
particle size distribution toward smaller sizes with the use of microwave
morphology [57,58], and an agglomeration of very small particles was
treatments. For example, ranges of 70–150, 60–130, and 40–90 nm were
observed. The spherical appearance of all samples is similar to that of
observed for samples B1 (40 W, 35 min), C1 (200 W, 35 min), and D1
commercial HAP derived from bovine bone (Merck) [59].
(400 W, 35 min), respectively. Furthermore, a narrow particle size dis­
The size and distribution of the particles were obtained by image
tribution (30–70 nm) was achieved with the highest microwave power
processing the SEM data. A trend of smaller particle size with increasing
and longest treatment times (Sample D3, 400 W, 45 min). There was a
microwave power and treatment duration, particularly at medium to
trend in particle size distribution toward smaller-sized particles with
high power levels (Samples C and D), is shown in Fig. 9. The growth rate
increased microwave power and extended irradiation time. Further­
and size of HAP crystals synthesized at low temperatures are affected by
more, the well-defined morphology of the in-demand HAP particles [65]
the supersaturation condition [60]. The saturation index and the degree
is represented by the narrow particle size distribution of the D3 samples.
of supersaturation of the reaction system are critical in the formation of
nanoHAP with different morphologies. This study was conducted at a
calcium precursor concentration of 1 M, which corresponds to the su­ 3.4. Hardness analysis
persaturation condition [61,62]. The SEM images revealed the forma­
tion of agglomerated spherical HAP, corroborating a previous study that The hardness of the nanoHAP was analyzed using a Vickers micro­
found that spherical nanoHAP was formed at a high saturation index, hardness test. According to Fig. 11, the hardness of the nanoHAP syn­
whereas nanoHAP with a nanorod or prism-like structure was formed at thesized without microwave treatment (Sample A) was 96 ± 18 MPa.
low saturation levels [63]. However, the study did not observe any The hardness increased at a microwave treatment power input of 450 W.
changes in HAP particle size at low temperature despite the supersatu­ Sample D1, with a 35-min microwave treatment realized an HV of 137
ration of the precursors. Additional energy introduced into the system ± 25 MPa. The highest HV of 345 ± 37 MPa was obtained from a 45-min
had no effect on particle size, as demonstrated by the low-power microwave treatment. Sample D3’s HV was comparable with that of

Table 3
Phosphate and hydroxyl vibrational modes in the samples.
Vibrational frequency (cm− 1)

Vibrational mode Ref 1 [51] Ref 2 [56] A B1 B2 B3 C1 C2 C3 D1 D2 D3

PO4 v1 963 962 962 962 962 962 962 962 962 962 962 962
PO4 v2 474 472 472 472 472 473 473 472 472 472 472
PO4 v3 1034 1032 1033 1032 1032 1031 1032 1033 1030 1032 1029 1033
PO4 v4 567 564 564 564 564 565 565 564 564 565 565
607 602 603 603 603 603 603 604 603 603 603 603
632 634 633 633 632 632
OH v1 3573 3572 3568 3568 3568 3568 3569 3570 3569 3570 3570 3569

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Fig. 8. Scanning electron microscopy images of nanoHAP synthesized without microwave treatment (A) and with microwave treatments (B, C, and D).

human cortical bones, which is in the range of 300–600 MPa for persons
aged 46–99 years [66]. Sample D3 exhibited a higher HV than that of
bovine bone sintered at 600 ◦ C (271 MPa), but it was lower than the
bone sintered at 750 ◦ C (588 MPa) [67]. A correlation between pro­
longed microwave irradiation (P < 0.05) and increased hardness was
noticed. We observed that prolonged microwave irradiation time tends
to reduce the crystallite size (Table 2) and the hardness was enhanced
with a smaller crystallite size. These observations are consistent with a
previous study that found a reciprocal relationship between hardness
and crystallite size in multi-ion doped hydroxyapatite [68].

3.5. Toxicity test

Although HAP is known as a biocompatible material, some studies

have reported its cytotoxic effects on human carcinoma cells [69,70].
The present study evaluated the cytotoxicity of HAP synthesized from
various processing parameters on human embryonic cells. High cell
viability after an incubation period of 48 h was demonstrated in all types
of nanoHAP, which indicates that the process parameters applied in this
Fig. 9. Average particle size of nanoHAP synthesized without microwave study did not produce HAP that was cytotoxic to human cells (Fig. 12).
treatment (A) and with microwave treatment (B, C, and D). The small letters on Furthermore, this result suggests that the HAP synthesized in this study
the chart indicate significant differences between samples (P < 0.05). has good biocompatibility with HEK293T cells, which is one of the
model cell lines used in the evaluation of the potential cytotoxicity of
materials for biomedical applications [71]. Some nanoHAP samples
demonstrated higher cell viability compared with the control, which
indicates the potential of nanoHAP to induce cell growth.

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Fig. 10. Particle size distribution of nanoHAP synthesized without microwave treatment (A) and with microwave treatment (B, C, and D).

4. Conclusion (Samples B1 and B2, 40 W) produced nanoHAP with similar properties

as the control. Increasing the power and irradiation time (Samples C and
This study successfully synthesized nanoHAP using eggshells, as a D) resulted in nanoHAP with smaller particle sizes (as depicted by the
calcium resource, at nine different conditions with varying microwave SEM histograms) compared with the nanoHAP from control. An
power and irradiation times. The complete formation of HAP was improved particle size homogeneity was achieved when the synthesis
confirmed by the disappearance of the CaO peak in the diffractograms of was conducted at high irradiation power (Sample D, 400 W). The crys­
all samples. The use of microwave energy was shown to induce the tallinity of the nanoHAP was observed to be influenced by the irradia­
synthesis of nanoHAP. The energy emitted by microwave heating was tion time. The nanoHAP crystallinity increased when the irradiation
confirmed to both improve the nucleation of nanoHAP and increase the time (at the same irradiation power) was increased, according to crys­
quantity of the precipitate produced. An SEM analysis confirmed the tallinity calculations using the XRD and FTIR (height) methods. Sample
presence of nanoHAP. A low microwave irradiation power and time D’s hardness test also revealed that irradiation time improves the

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