Results in Physics 17 (2020) 103051
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Results in Physics
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A comparative study of the mechanical integrity of natural hydroxyapatite T
scaffolds prepared from two biogenic sources using a low compaction
pressure method
E.S. Akpana, M. Daudaa, L.S. Kuburia, D.O. Obadaa,d,⁎, D. Dodoo-Arhinb,c,⁎
a Department of Mechanical Engineering, Ahmadu Bello University, Zaria, Nigeria
bDepartment of Materials Science and Engineering, University of Ghana, Legon, Ghana
c Institute of Applied Science and Technology, University of Ghana, Legon, Ghana
dAfrica Center of Excellence on New Pedagogies in Engineering Education, Ahmadu Bello University, Zaria, Nigeria
A R T I C L E I N F O A B S T R A C T
Keywords: With a view to enhancing laboratory and application-based pedagogical approaches in bioengineering, a com-
Sintering parison of the physical, chemical and mechanical properties of natural hydroxyapatite produced from non-
Mechanical properties separated animal bones (NB) and catfish bones (CB) obtained by thermal treatment and a low compaction
Apatite pressure method has been reported in this study. The structure, morphology and surface chemistry of the pro-
Bone implants cessed biomaterials were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and
Ca/P molar ratio
infrared spectroscopy, respectively. Uniaxial compaction using a pressure of 1 MPa was used on circular shaped
hydroxyapatite scaffolds to measure the mechanical properties of the produced scaffolds. From XRD analysis,
both samples showed prominent reflections of the hydroxyapatite phase, suggesting high crystallinity and phase
stability. The morphology of the powders showed irregular shapes with large agglomerates for non-separated
animal bones-derived hydroxyapatite as compared to more open pores in the catfish bones-derived hydro-
xyapatite. Hydroxyapatite produced from catfish bones revealed a microstructure with open pores which is
useful in terms of cell adhesion. The hydroxyapatite products revealed Ca/P ratios of 1.58 and 1.63 for catfish
bones (CB) and non-separated animal bones-derived hydroxyapatite, respectively. Improvements in the fracture
toughness were observed for CB in comparison with NB. Calculated fracture toughness values were 5.72
MPa. m1/2 and 2.35 MPa. m1/2 for catfish bones and non-separated animal bones-derived scaffold respectively.
These results are useful in terms of the production and biomedical applications of natural hydroxyapatite.
Introduction mechanical integrity may be linked to heat treatment which initiates
and allow the propagation of rough grained morphology, resulting in
It is well known that the production of hydroxyapatite (HAp) has poor mechanical characteristics. Mechanical properties evaluation and
consistently been processed from natural sources such as animal bones dataset is very important to ascertain the mechanical integrity of de-
[1], fish bone [2], coral [3], and egg shells [4–6]. Hydroxyapatite from veloped hydroxyapatite scaffolds. A plethora of studies to ascertain the
natural sources is preferable as compared to the synthetic type for mechanical characteristics of hydroxyapatite scaffolds is available in
biomedical applications [7]. This is because natural HAp has, in trace the literature [1,12–18], while there are inadequate mechanical prop-
amounts, magnesium, potassium, strontium and sodium inherent in its erty datasets that reveal comparisons for apatites produced from dif-
chemical structure. Hydroxyapatite (HAp) by virtue of its unique ferent natural sources.
characteristics is quite useful as medical implants and hard tissue re- The brittleness of bio-ceramic has over time limited their applica-
placement by virtue of its huge similarity with hard tissue of human tions in biomedicine as they exhibit strength when subjected to com-
bones and teeth [8–10]. pressive forces but quite weak under tensile and shear forces. It is
Nonetheless, the use of HAp is hampered mechanically by virtue of possible to enhance the mechanical properties of HAp by preparing the
its low fracture toughness, making it liable to failure [11]. The low scaffolds with relatively high compaction pressure. This protocol has
⁎ Corresponding authors at: Department of Mechanical Engineering, Ahmadu Bello University, Zaria, Nigeria (D.O. Obada). Department of Materials Science and
Engineering, University of Ghana, Legon, Ghana (D. Dodoo-Arhin).
E-mail addresses: doobada@abu.edu.ng (D.O. Obada), ddodoo-arhin@ug.edu.gh (D. Dodoo-Arhin).
https://doi.org/10.1016/j.rinp.2020.103051
Received 15 December 2019; Received in revised form 4 March 2020; Accepted 4 March 2020
Available online 07 March 2020
2211-3797/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license 
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
E.S. Akpan, et al. Results in Physics 17 (2020) 103051
Fig. 1. Methodological protocol for the synthesis and characterization of produced HAp.
conformed to progress made in the fabrication of bioceramics. As per
process parameters, to produce pure and clinically required hydro-
xyapatite, one facile method of producing hydroxyapatite scaffolds is
cold isostatic pressing. In this way, sintering after shaping of green
ceramic compacts has been done [1,19,20]. In order to improve the
homogeneity in densification and microstructure, cold pressing is viable
by virtue of its isotropic pressure. In addition, this protocol has been
used to manufacture biomaterials derived scaffolds and powders with
fewer pores [21]. Stemming from these, studies have reported a re-
duction in compressive strength when compaction pressure is applied.
This is possible because of the stress fields induced in the HAp powders
during compaction which has the tendency to make it more susceptible
to cracks. In a recent study by our group [1,20], very low cold com-
paction pressure (500 Pa) was proposed for compacting HAp powders,
and this produced promising mechanical properties to a large extent
due to reduced stress fields during powder compaction.
Here, we report a comparison of the physical, chemical and with
more emphasis, the mechanical characteristics of hydroxyapatite pro-
duced from non-separated animal bones and catfish bones. The me-
chanical properties of the sintered bioceramics scaffolds assisted by low
cold compaction pressure were presented and discussed.
Materials and method
Production of HAp powder from non-separated biowastes and catfish bones
Non-separated animal bones which are regarded as wastes were
obtained from an abattoir in Zaria, Nigeria, while the catfish bones
were sourced from restaurants, since they were tagged as wastes as
well, in Zaria metropolis. The sourced bones (raw materials) were used
as precursors for hydroxyapatite production. The non-separated animal
bones were thoroughly cleaned and a detailed procedure can be found
in studies by our group [1,20]. This procedure was used to clean/de-
proteinize the catfish bones as well. Tap water was used for cleaning the
bones throughout the process. The cleaned bones were burnt by a flame
in open air and subjected to sintering at 900 °C for 2 h at a heating rate
Fig. 2. X-ray diffraction patterns for the synthesized CB and NB-derived hy- of 5 °C/min. The sintered powdery hydroxyapatite samples sourced
droxyapatite and identification of the HAp lattice planes. from the non-separated and catfish bones were labelled as NB and CB,
respectively. These sample nomenclature were adopted in this study.
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E.S. Akpan, et al. Results in Physics 17 (2020) 103051
Table 1
Cell parameters of HAp derived from the biowaste bones.
Calcination temperature (oC) Sample Code Average crystallite size (nm) Crystallinity (%) c/a ratio
900 CB 37.2 99 0.79
900 NB 99 86.15 0.52
I
x = 100 × 300
− V112/300
c I300 (1)
where I300 represents the intensity of (3 0 0) diffraction peak, V112/300
represents the reflection of the hollow between (1 1 2) and (3 0 0)
diffraction peaks of HAp. Identification of phases was done by com-
paring the diffraction patterns of HAp with JCPDS standards
Fourier transform infrared (FT-IR) analysis
Surface chemistry data was collected on a Fourier Transform
Infrared Spectrometer at a resolution of four wavenumbers, operating
from 4000 to 400 cm−1.
Scanning electron microscopy (SEM) and energy dispersive X-Ray
spectroscopy (EDX) analysis
The microstructure of the samples was studied on a Phenom ProX
Desktop scanning electron microscope (SEM) equipped with EDX for
elemental mapping and operated at 15 kV. Each sample was viewed at
low magnifications of 300 and 500 xk. The EDX maps indicated the
Fig. 3. FT-IR spectra for the synthesized CB and NB derived hydroxyapatites. atomic and weight percentages of each element analyzed from obtained
HAp. To view the samples at higher magnification, images were taken
After sintering, the obtained HAp powders were milled in a metallic at 5000× using a JEOL JSM-7600F scanning electron microscope op-
mortar and passed through a sieve of 300 μm mesh prior to powder erated at 15 kV.
analyses. The methodological approach for the processing, physical and
mechanical properties measurement of the synthesized HAp is as shown Optical microscopy
in Fig. 1. (Note: CB and NB were separately processed following the Optical microscopy images obtained using an optical microscope
methodological sequence). ((ICC50, DM 750, Leica, Wetzlar, Germany) was used to reveal some
inherent structure of the produced apatites.
Chemical characterization of produced hydroxyapatite Physical characterization of produced hydroxyapatite
X-ray diffraction (XRD) analysis The physical characterization was split into two levels; measure-
To investigate the structure and phases present in the synthesized ment of porosity and the bulk powder densities
HAp powders (NB and CB), diffraction patterns were collected on a
Rigaku Miniflex diffractometer and powder crystallinity was calculated Porosity measurements
as expressed in Eq. (1) [22]: Porosity measurements of the produced hydroxyapatite were con-
ducted using the expression from Eq. (2):
Fig. 4. Scanning electron micrographs of CB and NB-derived hydroxyapatite at high magnification (×5000).
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E.S. Akpan, et al. Results in Physics 17 (2020) 103051
Fig. 5. Scanning electron micrographs of CB and NB-derived hydroxyapatite.
⎛ Weight of HAp ⎞ card number 09-0432, which contains sharp and prominent reflectionsPorosity(%) = ⎜1 − ⎟ × 100
⎝ Volume of HAp × Density of HAp⎠ (2) due to the high degree of crystallinity of the powders. The estimated
degree of crystallinity was around 99.9% and 86.15% for CB and NB,
The density of HAp used is the typical density of HAp (3.16 g/cm3) respectively, while the mean crystallite size of CB and NB taken from
[1,23–25]. 2θ = 31.72° (211 plane) was 37.1 nm and 99 nm, respectively. The
higher degree of crystallinity for CB means the derived hydroxyapatite
Density measurements is more stable as compared to NB. Sintering the powdery hydro-
The apparent densities of the produced hydroxyapatite were cal- xyapatite precursors resulted in degradation of the hydroxyapatite
culated from the dry weight and volume of the powders using the ex- phase to form small amounts of tetra-calcium phosphate (TTCP). The
pression from Eq. (3): presence of TTCP may not be detrimental since this phase is bio-
Dry weight of HAp compatible and has a high degree of solubility than hydroxyapatite
Apparent Density of HAp = [42].
Volume filled by HAp (3) Table 1 shows the crystallinity, crystallite size and cell parameters
of the heat treated hydroxyapatite sourced from the biowastes which
Mechanical testing were estimated from the X-ray diffraction data. The variations observed
in the crystallinity of the samples can be attributed to the gradient in
The powders were uniaxially pressed using a cold compaction molecular positioning during heat treatment. However, considerable
pressure of 1 MPa in a 25 mm diameter cylindrical die. The produced crystallinity was recorded for the samples. The crystallite sizes show an
green compacts were sintered in air atmosphere at 900 °C for a 2 h inverse relationship as compared to the crystallinity of samples. For CB
dwell time and at a heating and cooling rate of 5 °C/min. with comparative small crystallite size (37.2 nm), the crystallinity is
The mechanical properties measurement conducted for the fabri- higher and for NB with a large crystallite size (99 nm) the crystallinity
cated scaffolds were hardness, compression strength, elastic modulus, is comparatively smaller. It means that the crystallite size is not de-
yield strength, fracture toughness and brittleness index. The details of pendent on crystallinity and a small crystallite size can cause the
all mechanical evaluations are reported elsewhere [1]. broadening of the XRD peaks as observed on the XRD patterns for CB
(see Fig. 1). The values of the lattice parameters ratio (c/a) obtained in
Results and discussion this study are close to values obtained in a study carried out by Pal et al.
[41].
XRD diffraction analysis
Surface chemistry
The XRD patterns for the synthesized CB and NB derived HAp are
presented in Fig. 2. The major reflections matched those in the JCPDS The FT-IR spectra obtained from the CB and NB powders (Fig. 3)
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E.S. Akpan, et al. Results in Physics 17 (2020) 103051
Fig. 6. EDX spectra of CB and NB derived hydroxyapatite.
exhibited a range of bands from 1100 ± 1550 cm−1 in addition to the and NB particles presented layered shapes typical of HAp [26]. These
peaks expected for hydroxyapatite (i.e. those related to the PO 3−4 and results are in tandem with the SEM results obtained by [27] and [28]. It
OH− groups). Despite subjecting both HAp powders (CB and NB) to is worth noting that the images revealed the appearance of more open
similar processing protocol, there was a broad peak around 3417 cm−1 pores in the bulk of CB with lesser agglomerated structure, hence, the
in the spectra for NB, demonstrating the presence of adsorbed water pores became wider (larger). Micrographs for CB show interconnected
[20]. This broad band is attributed to the ν3 and ν1 stretching modes of (open) porosity, and linking the degree of pore connectivity to the
water molecules [43]. In addition, the band observed at 3497 cm−1 apparent density (Table 1), there are some trends evident in the var-
indicates the characteristic of O–H stretching vibration of HAp [44]. iation of micro pore size and morphology, with apparent density. As
Bands that are similar to those obtained in this study have also been expected, the micro pore sizes were found to decrease with increasing
observed in previous studies [43,45,46]. The inclusion of the bands of apparent density. It is also noted that the micropores were isolated
carbonate in NB is attibuted with the adsorption of non-structural spherical pores, which were randomly distributed throughout the bio-
carbonate on the surface of NB [47]. ceramic space for CB and NB and located within the ceramic grains. The
higher porosity of prepared CB as compared to NB (Table 1) can be
SEM/EDX analysis advantageous for applications in biomedicine.
EDX analysis revealed the elemental composition and the corre-
The microstructure of CB and NB powders at high magni cation is sponding Ca/P molar ratio of the HA powders as shown in Fig. 6. Thefi
shown in Fig. 4. Typical ower-like microstructure which consists of main elements of CB and NB are calcium (Ca), phosphorus (P) andfl
akes of petal-like orientation are visible for CB derived hydro- oxygen (O) with an average atomic Ca/P molar ratio of 1.58 and 1.63fl
xyapatite. Flower like HA morphology has been described as an e - for CB and NB respectively. The EDX analysis reveals that HA extractedffi
cient carrier for the delivery of drugs [48].Rod-shaped particles are from the produced hydroxyapatite (CB and NB) contains ions such as
evident for NB and this description is similar to reports elsewhere [49]. Na
+, Mg2+ etc. in trace amounts such as reported by [29] and [30].
The orientation of the NB particles which are inter-connected with Presence of these trace ions are advantageous for natural HAp. These
grain structures which are close, typically de nes the initiation of a trace ions have the potential to enhance the formative and regenerativefi
defined crystalline grain structure of hydroxyapatite. processes in bone [31].
SEM images at low magni cations, ×300 and ×500 for CB and NB The Ca/P ratios of HAp obtained in this study has slight variationsfi
is presented in Fig. 5 and the grain distribution and orientations are from the theoretical value for stoichiometric HAp of 1.67. One factor
observed. As noticed, the surface of CB and NB is overly porous and this variation is attributed to, is the processing (burning in open air),
there is non-uniformity in the particle size distribution. At both mag- which may not have completely removed the fats and soft tissues and
ni cations, the powders are irregular in shape with more agglomerates possibly have increased the elemental composition of carbon (C) in thefi
noticed for the NB powders and quite smaller pores are present. The CB resultant powders after sintering. Furthermore, the sintering
5
E.S. Akpan, et al. Results in Physics 17 (2020) 103051
Fig. 7. Optical micrographs of NB and CB samples.
Table 2
The densities of CB and NB-derived hydroxyapatite.
Samples Notation %D1 %D2 %D3 %Davg SD
CB 1.63 1.60 1.62 1.62 0.01
NB 1.69 1.69 1.67 1.68 0.01
% D1-3 = Percentage densities of the 2 samples.
SD = Standard Deviation.
Table 3
The porosities of CB and NB-derived hydroxyapatite.
Samples notation %P1 %P2 %P3 %Pavg SD
CB 48.4 49.2 48.7 48.8 0.33
NB 46.7 46.5 47.2 46.8 0.03
% P1-3 = Percentage porosities of the 2 samples. Fig. 8. Hardness and compressive strength values of CB and NB derived scaf-
SD = Standard Deviation.
folds.
temperature which influences the composition of compounds with in-
herent calcium could be another reason for this deviation as reported in which can be traced to the high carbon content observed in the EDX
studies elsewhere [29,32,33]. In similar vein, the high carbon content spectra of the produced HAp. The images for CB and NB show much
from EDX analysis can be attributed to high cholesterol and protein diversified walls as per internal and external shapes and orientation.
concentrations and the calcium hydroxyapatite rich areas. These results For the deproteinized CB and NB samples, dots of fats can be observed
are similar to a study conducted by [34], where they inferred that showing residual matter and this can also be observed for the sintered
cholesterol-rich areas of calcium hydroxyapatite showed higher signals samples as well. This may have caused the high carbon content as re-
for C and O and also, areas rich in calcium showed greater amounts of vealed in the elemental composition (see Fig. 6) and the formation of
C, O, P, and Ca. three dimensional carbonaceous structures observed in the optical mi-
The optical microscopy images as shown in Fig. 7 were used to crostructures of the sintered samples.
examine the passage of light through the powders in order to reveal its
internal morphology. Optical microscopy observations show a texture
6
E.S. Akpan, et al. Results in Physics 17 (2020) 103051
in the eventual application of these ceramics, that the compressive
strength of CB was no higher than 2 MPa, a value that can be considered
sufficient with room for improvement. Also, the main advantage of
these produced bio-ceramics, beyond their capability of providing di-
versified architectures and a variety of pore size distribution, is the fact
that they have satisfactory mechanical strength - allowing for use under
less load bearing conditions.
Conclusions
In this study, catfish bones and non-separated animal bones-derived
HAp has been produced and a comparison of the physico-mechanical
properties has been reported. Therefore, the following conclusions can
be reached:
1. From the phase analysis of the produced hydroxyapatites, a domi-
nant phase of HAp was observed with reflections of tetra-calcium
Fig. 9. Young modulus and fracture toughness values of CB and NB derived phosphate, which is also a biocompatible phase.
scaffolds. 2. The higher porosity of prepared catfish bones-derived hydro-
xyapatite as compared to the non-separated animal bones-derived
Samples density and porosity measurements hydroxyapatite (Table 2) can be useful for applications in biome-
dicine
Apparent density (Table 2) for CB and NB was found to range from 3. The hardness values of the synthesized hydroxyapatites are in the
1.62 ± 0.01 and 1.68 ± 0.01 g/cm3 respectively which depicts that range of human cortical bone.
NB is denser with less open porosity. Table 3 reveals the variations in 4. The specific mechanical properties of the samples were related to
porosity for CB and NB. The porosity of CB, 48.8 ± 0.33% is higher as the calculated apparent densities of the samples with the compres-
compared to NB which was calculated as 46.8 ± 0.03%. It has been sive strength increasing from approximately 0.47 to 1.92 MPa for
reported that porosities in the range of 40 to 90% encouraged os- non-separated animal bones-derived hydroxyapatite and catfish
teointegration [1,35]. It is safe to say that the calculated porosities bones-derived hydroxyapatite, respectively. This advances the via-
show potential of the suitability of CB and NB for biomedical applica- bility of catfish bones-derived hydroxyapatite in this regard for use
tions. Stemming from this, CB has greater potential to be used for in biomedical applications where compressive stresses are a
biomedical applications where higher porosity would be useful. common phenomenon.
Mechanical measurements CRediT authorship contribution statement
The Vickers hardness and compression strength variation is shown E.S. Akpan: Formal analysis, Writing - original draft. M. Dauda: .
in Fig. 8. Vickers hardness increased from a value of 0.48 GPa for CB to L.S. Kuburi: Writing - review & editing. D.O. Obada: Formal analysis,
a value of 0.65 GPa for NB. The increase in hardness could be attributed Writing - original draft, Writing - review & editing. D. Dodoo-Arhin:
to the increase in relative density as shown in Table 3. It is well known Writing - original draft, Writing - review & editing.
that the hardness of HAp scaffolds increase with increasing relative
density [48]. The relative decrease in the Vickers hardness of CB can Declaration of Competing Interest
also be attributed to the decomposition of HAp phase [50,51] as con-
firmed by the formation of small amounts of tetra-calcium phosphate The authors declare that they have no known competing financial
(TTCP) as shown in Fig. 2.The variation in compressive strength can be interests or personal relationships that could have appeared to influ-
related to the calculated apparent densities of the samples with the ence the work reported in this paper.
compressive strength increasing from approximately 0.47 to 1.92 MPa
for NB and CB respectively This advances the viability of CB in this References
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