Separation and Purification Technology 161 (2016) 129–135

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Separation and Purification Technology

journal homepage: www.elsevier.com/locate/seppur

Extraction of biocompatible hydroxyapatite from fish scales using novel

approach of ionic liquid pretreatment
Nawshad Muhammad a,b,⇑, Yanan Gao a,⇑, Farasat Iqbal b, Pervaiz Ahmad c, Rile Ge a, Umar Nishan d,
Abdur Rahim b, Girma Gonfa e, Zahoor Ullah e
a
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China
b
Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore, Pakistan
c
Department of Physics, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
d
Department of Chemistry Kohat University of Science and Technology, Kohat, KPK, Pakistan
e
Petronas Ionic Liquid Centre, Department of Chemical Engineering, Universiti Teknologi Petronas, 31750 Tronoh, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: In this study the waste fish scales (FS) were dissolved in 1-butyl-3-methylimidazolium acetate ionic liq-
Received 29 September 2015 uid to obtain a valuable product of hydroxyapatite (HAp). The HAp was obtained in the yield of 32 ± 2%.
Received in revised form 27 January 2016 The obtained HAp was characterized using Fourier Transform Infrared Spectroscopy (FTIR), Powder X-
Accepted 30 January 2016
rays Diffraction (PXRD), Thermal Gravimetric Analysis (TGA), Field Emission Scanning Microscopy (FE-
Available online 4 February 2016
SEM), Energy Dispersive X-rays spectroscopy (EDX), and Brunauer–Emmett–Teller (BET). The results of
FTIR and XRD showed the characteristic peaks of the HAp. The thermal degradation temperature of the
Keywords:
extracted HAp was relatively high. Furthermore, low weight loss was measured which confirmed the
Ionic liquid
Fish scale
removal of organic part of FS during ionic liquid treatment. The FE-SEM result showed the particles with
Hydroxyapatite different morphologies and EDX analysis showed a Ca/P ratio of 1.60 for the extracted HAp. The biocom-
Extraction patibility of the extracted HAp was assessed through MTT cell viability assay using known Human
Characterization Embryonic Kidney 293 cells (HEK cells) and epidermoid carcinoma cells (A431 cells).
Biocompatibility Ó 2016 Elsevier B.V. All rights reserved.

1. Introduction
It has been approximated that each year 18–30 million tons of
fish waste is discarded which accounts for 50% of total mass of fish
processing industry in the world. In the fish waste, the fish scale
was estimated 4% by weight [1,2]. Fish scales consist mainly of col-
lagen and hydroxyapatite [HAp] along with fatty acids, vitamins,
antioxidant and trace elements [2,3]. Collagen is an abundant pro-
tein that has many applications in biomedical and pharmaceutical
sciences [4]. HAp [Ca10(PO4)6(OH)2] having similar properties with
natural bones, has gained immense importance in the field of
orthopedics and in the development of dental materials [5,6].
HAp has been synthesized by various chemical methods like solid
state reaction, hydrothermal reaction, co-precipitation reaction
and sol–gel synthesis mechano-chemical methods [7–10]. The
laser ablation technique was used to fabricate the HAp based
⇑ Corresponding authors at: Interdisciplinary Research Center in Biomedical
Materials, COMSATS Institute of Information Technology, Lahore, Pakistan.
E-mail addresses: nawshadchemist@yahoo.com (N. Muhammad), ygao@dicp.ac.
cn (Y. Gao).
nanoparticles while their thin film were coated using the electro-
static spray and aerosol deposition techniques [11–13].
HAp has also been extracted from various biological sources like
bones and fish scales, etc. [14,15]. All these methods involve the
use of acids, alkalis and heat to separate the HAp from biological
sources. Along with environmental issues related to the use of
acids and alkalis, the use of high temperature during the course
of treatment also results in distortion of natural intact physical
structure of the extracted HAp. In addition, the use of all these
methods results in loss of other major constituents: collagen which
has been acknowledged for various applications.
It is well documented that ionic liquids, due to their advanta-
geous properties (negligible vapor pressure, nonflammability, non-
explosiveness, electrochemical and thermal stability, easy
recyclability and high conductive characters), has gained impor-
tance for the last 25 years in many fields. Likewise, ionic liquids have
been reported for the dissolution of other biopolymer, like cellulose,
chitosan, chitin, collagen and silk, etc. [16–20]. The novelty of this
work lies in the use of ionic liquid first time for extraction of HAp
from fish scales. In this study the waste fish scales were collected
from the local market and dissolved in 1-butyl-3-
methylimidazolium acetate ionic liquid to obtain the HAp.

http://dx.doi.org/10.1016/j.seppur.2016.01.047
1383-5866/Ó 2016 Elsevier B.V. All rights reserved.
130 N. Muhammad et al. / Separation and Purification Technology 161 (2016) 129–135

Comparatively, the fish scales was preferred to fish bones, as the

bone matrix is hard and difficult to bring into small size particle as 872
C 1637 1418
it needs more crushing and ball milling. Fish scales consist of more
collagen as compare to fish bones that has been targeted to separate 872
simultaneously with extraction of HAp during ionic liquid pretreat-

Transmittance (%)
A 1240
ment. The collagen part is compiled as separate study and will be
1418
published elsewhere. The HAp was separated from the resultant 1544
mixture and its characterization was performed using FTIR, FE-
1640
SEM, XRD, TGA, particle size distribution and BET surface analyzer.
The biocompatibility of extracted HAp was assessed through the cell
viability assay using the known cells of HEK and A431. 872
B 600559
1640
2. Experimental 1418
3433
1039
2.1. Materials and methods
4000 3500 3000 2500 2000 1500 1000 500
Carp fish (Cyprinidae) was obtained from local market located Wavenumber/cm-1
opposite to Dalian Institute of Chemical Physics, Chinese Academy
Fig. 1. FTIR spectra of fish scale (A), extracted hydroxyapatite (B) and commercial
of Sciences in Dalian, China. FS was isolated from the fish and
synthetic hydroxyapatite (C).
washed thoroughly with tap water followed by distilled water to
ensure the removal of undesired debris attached during scales
removal from fish. After washing, the FS was dried at room temper-
(a) Fish Scale
ature and then ground with grinder (AISITE, China). 10.0 g of 1-
(b) HAp
butyl-3-methylimidazolium acetate (CJC, China) was charged into
(c) Ref. 900-3550
30 mL reagent bottle followed by addition of 0.5 g of ground fish
scales. The FS was added in two fractions during the course of pre-
(a)
treatment. After charging, the bottle’s cape was closed and kept in
Intensity (a.u.)

the oil bath and heated for 12 h at 100 °C. After dissolution process,
water of equal volume of reaction mixture was added followed by

(002)
addition of NaOH solution (0.5 M) to bring the pH at 9. The HAp
(b)
was obtained as precipitate by centrifugation (11,000 rpm) for

(112)
30 min. The supernatant contained the ionic liquid and other con-
(210)

(213)
stituents of FS were further subjected to fractionation and ionic liq-

(222)
(310)

(004)
uid was recycled. The supernatant contained the ionic liquid,
organic portion etc. was collected and NaCl solution (2 M) was
added followed by adding HCl solution (0.5 M) to bring the pH
around 2. The precipitate of organic portion (collagen) was (c)
obtained which has been separated from the resulting mixture
by centrifugation and subsequently washing at 11,000 rpm. The 10 20 30 40 50 60
resultant acidic supernatant was neutralized with NaOH solution 2-Theta (degree)
(0.5 M) and further subjected to rotary evaporator to remove the
Fig. 2. XRD analysis of fish scale (a), extracted hydroxyapatite (b) and hydroxya-
water. Ionic liquid was extracted with acetone and dichloro- patite Reference card 9003550 HAp (c).
methane followed by subsequent filtration and evaporation.

2.2. Characterization 110

FS
FT-IR spectrometer (Bruker Tensor 27) was used to characterize 100
(a) HAp
the sample. The samples were prepared as a compressed KBr discs
90
and measurements were performed in the range of 450–
4000 cm
1. Powder XRD (X-rays diffraction model; PANalytical
Weight loss (%)

80
X’Pert Pro Multi-Purpose Diffractometer) was used to confirm the 100 Magnified TGA spectra of extracted HAp

phase composition of extracted HAp. The XRD spectra were 70 (b)

Weight loss (%)

recorded from 5° to 80° and step size of 0.1°. Thermal behavior 95

of the samples was analyzed using thermal analyzer (NETZSCH, 60

90
STA 449) over a temperature range of 42–780 °C and heating rate
50
of 10 °C min
1 in nitrogen atmosphere. The morphology of the 85
samples was investigated using Field Emission Scanning Micro- 40
0 200 400 600 800 1000
Temperature/°C
scopy (FE-SEM, Jeol JSM-7800F, Japan). Energy dispersive X-rays
spectrometry (EDX) was used for elemental analysis. The particle 30
size distributions were measured using Zetarsizer nanoseries (Mal-
0 100 200 300 400 500 600 700 800 900 1000
vern Ltd., UK). For particles size distribution measurement, 0.02 g
of extracted HAp was dispersed in the 2 mL of ethanol to make a Temperature/°C
1% solution. Brunauer–Emmett–Teller (BET) method was used to Fig. 3. TGA of fish scale (FS, black; a) and extracted hydroxyapatite (HAp, blue; b).
measure the surface area for the samples at 77 K of liquid N2. Prior (For interpretation of the references to color in this figure legend, the reader is
to adsorption and desorption of N2 gas, the samples were degassed referred to the web version of this article.)
 N. Muhammad et al. / Separation and Purification Technology 161 (2016) 129–135 131

(FS) (HAp)
Fig. 4. FE-SEM micrograph of fish scale (FS), extracted hydroxyapatite (HAp).

Table 1 in a 96-well plate overnight and treated with various concentra-

Elemental analysis of fish scale (FS), extracted hydroxyapatite (HAp). tions of each type of HAp (125 lg mL
1, 62.5 lg mL
1 and
Element App Con. Weight % Atomic % 31.25 lg mL
1) for 24, respectively. 10 lL of MTT stock solution
FS HAp FS HAp FS HAp
(5 mg/mL) was added to each well, and incubated for a further
4 h. After careful removal of the medium, 100 lL dimethyl sulfox-
CK 1.04 2.93 5.52 2.40 13.67 5.35
ide (DMSO)-isopropanol (1:1) was added to each well, and the
NK 0.16 0 3.20 0 6.80 0
OK 1.10 12.16 5.49 24.75 10.21 41.39 plate was then shaken until the crystals were solubilized. Finally,
PK 5.95 11.57 25.93 23.63 24.90 20.41 the absorbance was measured at 540 nm by Microplate Spec-
Ca K 8.81 15.43 59.85 49.22 44.41 32.85 trophotometer (Synergy H1, serial No. 265511). The cell survival
percentage was calculated as OD treatment/OD control  100%,
where OD is optical density. The OD treatment is related to sample
under vacuum by heating at 393.15 K in Master Prep instrument. and OD control related to positive control i.e. Cis-platinum.
The measurement was performed on Quanta-chrome autosorb-1
instrument (USA). The average pore size and pore volume were
determined at relative pressure P/Po = 1, while BET equation was 3. Results and discussion
used to calculate the surface area of the samples.
Ionic liquids with high hydrogen bond basicity values are
known to dissolve the biopolymer like cellulose by interrupting
2.3. Cell viability the hydrogen bond linkage in their constituents [21]. FS consists
of collagen matrix in which HAp is embedded inside the lattices
The influences of extracted HAp and commercial HAp (CAS No. or on surface [22]. During pretreatment with ionic liquid, the col-
1306-06-5, Aladdin chemical Co (shanghai)) on cell viability were lagen part interacted with ionic liquid through hydrogen bonding
determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte which caused its dissolution [19] while the inorganic part, HAp,
trazolium bromide (MTT) assay. The samples were diluted in HBSS remained undissolved which has been obtained as a precipitate.
buffer and freshly diluted in culture medium before experiment. The 1-butyl-3-methylimidazolium acetate having high hydrogen
HEK-293 cells or A431 cells (5  105 mL
1, 100 lL) were cultured bond basicity value (b = 1.18) and dissolution capacity was

(FS) (HAp)
Fig. 5. EDX analysis of fish scale (FS), extracted hydroxyapatite (HAp).
132 N. Muhammad et al. / Separation and Purification Technology 161 (2016) 129–135

Table 2
Positive control Commercial HAp Extracted HAp
Surface area, pore volume and average pore size of extracted HAp and commercial 120
synthetic HAp.

Material BET surface area Pore volume Average pore size

100
(m2 g
1) (cm3 g
1) (nm)
HA 37.38 0.09 9.97

Viability (%)
a
CHAp 56.77 0.19 13.60 80
a
Commercial synthetic hydroxyapatite.
60
Quantity adsorbed and desorbed (cm3/g), STP

40
60
N2 adsorbed
N2 desorbed 20
50

40 0
1000 500 250 125 62.5 31.2
Concentration ( µ g/mL)
30
Fig. 7b. MTT assay of A431 cells incubated for 24 h.
20

10 3.1. FTIR analysis

0
0.0
Fig. 6. N2 adsorption isotherm of extracted hydroxyapatite (HAp).
selected for dissolution of FS [23]. The heating temperature was
maintained at 100 °C in order to minimize the effect of moisture
content as water decrease the dissolution ability of ionic liquid
through its hydrogen bonding interaction. The sample mixture
was heated for 12 h in order to make sure the complete dissolution
of the charged sample. After dissolution of organic component of
FS, the HAp was obtained as a precipitate with yield of 32 ± 2%
for the ground FS (the ground fish scales do not have the same con-
tent of HAp as unprocessed FS and it is assumed that some of the
HAp is lost during grinding). It has been observed that, while
increasing the pH of solution from 7 to 9, the yield of HAp
increased from 20 ± 2% to 32 ± 2%, however further increase in
pH was noted to have no such effect on the yield of extracted HAp.
120
Positive control
100
80
Viability (%)
Fig. 1 shows the FTIR spectra of FS, extracted HAp and commer-
0.2 0.4 0.6 0.8 1.0 cial synthetic hydroxylapatite (CAS No. 1306-06-5, Aladdin chem-
ical Co; Shanghai) in the range from 450 to 4000 cm
1. The peak
Relative pressure, P/P0
around 3433 cm
1 and 1640 cm
1 are assigned to the stretching
and bending vibration of OH group of adsorbed water [24]. The
amide II of collagen has vibration band around 1544 cm
1 which
is present in the FS whereas in the extracted HAp it is absent
[19]. The bands at 1418 and 872 cm
1 assigned to the asymmetric
stretching and out of plan bending mode of carbonate group (CO
23 )
are more obvious in the extracted HAp spectra [25]. The bands at
1251 cm
1 related to carbonate group CO
2 3 while the band at
1240 cm
1 assigned to amide III of collagen skeleton. The more vis-
ible bands at 1039, 600 and 559 indicate the asymmetric stretch-
ing, and symmetric and asymmetric bending vibrations of
phosphate group (PO
3 4 ) of extracted HAp and commercial syn-
thetic hydroxylapatite [25].
3.2. Powder XRD analysis
The XRD patterns of FS, extracted HAp and reference HAp are
shown in Fig. 2. The FS has a broad peak at 2h value of 22° along
with small shoulder peak at 2h value of 32.2° which corresponded
to the collagen biopolymer and HAp respectively [19,26]. The
Commercial HAp Extracted HAp extracted HAp consist of one phase which has been realized by
comparing with standards compiled by Crystallography Open
Database (COD) with hexagonal hydroxyapatite reference card
No. 9003550 (Fig. 2) with chemical formula of Ca10(OH)2(PO4)6.
In the extracted HAp, along with small peaks a well resolved char-
acteristic peak is observed at 2h value of 32.2° which corresponded

to the 211 plane of geometry [26]. The peaks of extracted HAp are
60 broader as compared to simulated standard spectra for hydroxya-
patite. The peak broadness was assumed due to either smaller
crystallite size or the amorphous nature of materials. Similar nat-
40
ure of broad peaks in XRD pattern by Huang et al. [25] for FS based
hydroxyapatite was reported and compared with hydroxyapatite
20 card (JCPDS 00-009-0432).

3.3. Thermal gravimetric analysis

0
1000 500 250 125 62.5 31.2
Concentration (µ g/mL) The thermal degradation behavior of FS and extracted HAp, and
their derivatives are shown in Fig. 3. The first weight loss (8%) was
Fig. 7a. MTT assay of HEK cells incubated for 24 h. observed at temperature less than 200 °C for both FS and extracted
 N. Muhammad et al. / Separation and Purification Technology 161 (2016) 129–135 133

HAp that corresponded to adsorbed water [27]. The second maxi-
mum weight loss for FS was observed at temperature of 335 °C
which is assigned to the decomposition of organic compounds
especially to collagen constituents. From 200 to 500 °C of temper-
ature, 58% weight loss was measured. In the case of extracted HAp,
a small weight loss was observed at 350 °C which accounts for 4%
from 200 to 500 °C that is related to the decomposition of tightly
attached organic compounds of the inner parts of the scale struc-
ture [28]. The decomposition at later stage was related to the
decarbonization of CaCO3 to form CaO for both FS and extracted
HAp [29,30]. The residues weighed at 780 °C were 30% and 87%
for FS and extracted HAp respectively which corresponds to the
biological apatite.
3.4. FE-SEM analysis
Fig. 4 shows the micrograph of FS and extracted HAp. The
obtained fish scales while grinding were changed into materials

A B

C D

Fig. 8a. Optical microscopy (10) of HEK cells (A), HEK_Commercial HAp (B), HEK_Extraxted HAp (C) and HEK_Cis-platinum (D) incubated for 24 h.

A B

C D

Fig. 8b. Optical microscopy (10) of A431 cells (A), A431_Commercial HAp (B), A431_Extraxted HAp (C) and A431_Cis-platinum (D) incubated for 24 h.
134 N. Muhammad et al. / Separation and Purification Technology 161 (2016) 129–135
of fibrous nature that contained debris on its surface as shown in
the Fig. 4. The extracted HAp shows particles of different morphol-
ogy. It consists of nano-scale particles along with microscale parti-
cles which have also been confirmed by the broad peaks exhibited
in the XRD pattern of Fig. 2.
3.5. EDX analysis
Fig. 5 and Table 1 shows the EDX analysis of FS and extracted
HAp which consisted of carbon (C), Nitrogen (N), Oxygen (O), Phos-
phorous (P) and Calcium (Ca) atoms. Comparatively, the extracted
HAp did not contain N and also the carbon content is lower to FS
which confirm the removal of organic components (collagen) dur-
ing ionic liquid pretreatment. The percentage atomic ratios of Ca/P
for FS and extracted HAp are 1.78 and 1.60 which is around 1.67
ratios measured in human bones respectively [31]. The results
show that extracted HAp deficient in Ca in the form of Calcium
phosphate (CaPO4) and Calcium carbonate (CaCO3).
3.6. Particle size distribution
The extracted HAp particles measured by Zetasizer particle ana-
lyzer showed a size distribution in the range of 1050–2900 nm
size. The distribution is mono-modal type with an average particle
size of 1870 nm. As the average particle size for extracted HAp
exceeds 100 nm thereby it is rendered as microcrystalline HAp.
3.7. BET surface analysis
Table 2 shows the values of BET surface area, pore volume and
pore size. The values measured for BET surface area, pore volume
and pore sizes are 37.38 m2 g
1, 0.09 cm3 g
1 and 9.97 nm respec-
tively of extracted HAp. The values obtained for extracted HAp are
slightly lower to the commercially available hydroxapatite. Pores
size are generally classified into various group sizes such as micro-
pores (<2 nm diameter), mesopores (2–50 nm) and macropores
(>50 nm). In light of the above, the pore size of the extracted
HAp is 9.97 nm which render them in mesoporous class of materi-
als. Fig. 6 shows that extracted HAp has significant surface area
which can be owed to use in different adsorption process especially
in drug loading for treatment of bone related diseases. The average
particle size was calculated 50 nm and 33 nm for the extracted
HAp and commercial synthetic hydroxyapatite respectively using
the following equation [32].
D ¼ 6=qSBET
where D is the average particle size in lm, q is the density of pure
HA (3.16 g cm
3), SBET is the BET surface area (m2 g
1).
3.8. Cell viability assay
The MTT assay on HEK cells and A431 cells for commercial HAp
and extracted HAp are shown in Figs. 7a and 7b. The Figures show
that irrespective of concentrations of both commercial HAp and
extracted HAp, the cell viability is more than 100%. While for cis-
platinum (positive control) the cell viability is less than 6%. It is
well documented that MTT assay is based on the capacity of the
mitochondrial enzyme, succinate-dehydro-genase of viable cells
to transform the MTT tetrazolium salt into a blue colored product,
MTT formazan and is proportional to the number of living cells
present [6]. The assay is used primarily in mammalian cell studies
as a measure of cell activation [33], cell growth and survival
[34,35] and to determine the chemosensitivity of cell lines [36].
The commercial HAp and extracted HAp, both shows higher cell
viability for A431 cells than HEK cells. The Figs. 8a and 8b show
the optical microscopic morphology of the cultured media in the
wells of microplate reader with or without addition of commercial
HAp, extracted HAp and cis-platinum (positive control) at concen-
tration of 31.2 lg mL
1 and incubated for 24 h. Fig. 8a shows the
even dispersion of HEK cells after addition of both commercial
HAp and extracted HAp while in case of cis-platinum addition
the cells are agglomerated which owed to the denaturing of living
cells. Similarly, the Fig. 8b shows agglomerated A431 cells for cis-
platinum added sample. This result corroborates with previous
work reported by Huang et al. which showed that fish scale based
HAp exhibit good cell viability [25]. In brief, the high cell viability
of HAp extracted from FS through ionic liquid pretreatment con-
firms its biocompatibility.
4. Conclusion
The 1-butyl-3-methylimidazolium acetate ionic liquid was
observed to dissolve completely the organic portion of FS while
the inorganic portion (HAp) was obtained as an insoluble material
in the yield of 32 ± 2. The characteristic peaks of HAp assigned to
phosphate (PO
2
2
4 ) and carbonate group (CO3 ) were identified in
the FTIR spectra. The XRD analysis of extracted HAp shows charac-
teristic peaks assigned to the hexagonal hydroxyapatite compiled
by International Center for Diffraction Data (ICDD). Comparatively,
low weight loss was measured for extracted HAp during thermal
gravimetric analysis. The FE-SEM analysis of extracted HAp
showed particles of different size and shapes. The percentage
atomic ratio of Ca/P for extracted HAp was 1.60 which is slightly
lower than the 1.67 ratios measured in human bones. The average
particle size measured, was 1870 nm for extracted HAp. The BET
surface area, pore volume and pore sizes were measured
37.38 m2 g
1, 0.09 cm3 g
1 and 9.97 nm respectively for extracted
HAp. The extracted HAp was assessed for its biocompatibility and
showed more than 100% cell viability for both HEK and A431 types
of cell lines.
Acknowledgment
This work has been supported by National Natural Science
Foundation of China (21273235).
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