Author’s Accepted Manuscript

Green Synthesis of Biocompatible Nanostructured

Hydroxyapatite from Cirrhinus mrigala Fish Scale
– A Biowaste to Biomaterial

Swamiappan Sathiskumar, Sekar Vanaraj, Devaraj

Sabarinathan, Somasundaram Bharath, Ganesan
Sivarasan, Subramanian Arulmani, Kathirvel
Preethi, Vinoth Kumar Ponnusamy www.elsevier.com/locate/ceri

PII: S0272-8842(19)30089-6
DOI: https://doi.org/10.1016/j.ceramint.2019.01.086
Reference: CERI20541
To appear in: Ceramics International
Received date: 4 January 2019
Revised date: 10 January 2019
Accepted date: 10 January 2019
Cite this article as: Swamiappan Sathiskumar, Sekar Vanaraj, Devaraj
Sabarinathan, Somasundaram Bharath, Ganesan Sivarasan, Subramanian
Arulmani, Kathirvel Preethi and Vinoth Kumar Ponnusamy, Green Synthesis of
Biocompatible Nanostructured Hydroxyapatite from Cirrhinus mrigala Fish
S c a l e – A Biowaste to Biomaterial, Ceramics International,
https://doi.org/10.1016/j.ceramint.2019.01.086
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Green Synthesis of Biocompatible Nanostructured Hydroxyapatite
from Cirrhinus mrigala Fish Scale – A Biowaste to Biomaterial

a,b*
Swamiappan Sathiskumar , Sekar Vanaraja, Devaraj Sabarinathana,
c d b
Somasundaram Bharath , Ganesan Sivarasan , Subramanian Arulmani , Kathirvel
a* b,d*
Preethi , Vinoth Kumar Ponnusamy

a
Department of Microbial Biotechnology, Biopharmacy lab, Bharathiar University, Coimbatore-

641046, Tamil Nadu, India.

b
Research Center for Environmental Medicine, Kaohsiung Medical University,

Kaohsiung City-807, Taiwan.

c
Department of Biotechnology, Translational Research lab, Bharathiar University,

Coimbatore-641046, Tamil Nadu, India.

d
Department of Medicinal and Applied Chemistry, Kaohsiung Medical University,

Kaohsiung City-807, Taiwan.

sathiskumarswamiappan@gmail.com

gunpre77@gmail.com

kumar@kmu.edu.tw

1
 *
Corresponding authors. Department of Microbial Biotechnology, Bharathiar University,
Coimbatore, Tamilnadu, India. Tel.: 08015273977.

ABSTRACT

In this study, we report a green approach to prepare biocompatible nanostructured

hydroxyapatite (NHAp) crystalline powders from Cirrhinus mrigala fish scale wastes by a

simple alkaline heat treatment process. The crystallinity and functional groups of the as-prepared

material were confirmed using X-ray diffraction and fourier-transform infrared spectroscopy.

The surface morphology was characterized using transmission electron microscopy. The

formation of NHAp and Calcium/Phosphorus (Ca/P) molar ratio was confirmed using Energy-

dispersive X-ray spectroscopy and Ca/P molar ratio was 1.67. The thermal stability of NHAp

was examined by thermogravimetric analysis. Further, the biocompatibility of as-synthesized

NHAp was examined towards the cell viability of MG-63 cells (human osteosarcoma cells) with

various dosages. NHAp boosts up the growth of MG-63 cells which showed superior cell

viability and ALP activity when compared with commercial HAp (CHAp). This result illustrates

that NHAp synthesized from Cirrhinus mrigala fish scale biowaste showed excellent

biocompatibility, and it can be a potential alternative biomaterial for various biomedical

applications.

Keywords: Nanostructured Hydroxyapatite, Biocompatibility, MG-63 cells, Cirrhinus mrigala

fish scales.

2
1. INTRODUCTION

Calcium phosphates (CP) are biomaterials consists of Calcium (Ca) and Phosphorus (P)
in a various molar ratio between 0.5-2.0 [1, 2]. Among them, hydroxyapatite
[(Ca)10(PO4)6(OH)2; HAp] is a major CP-based biomaterial which mimics mineral element
possessed in natural teeth and bones [3,4]. Among them, HAp containing Ca/P molar ratio of
1.67 exhibits remarkable bioactivity [5] and biocompatibility [6,7] in the biomedical
applications. It has been used as a dental implant [8] and bone substitute material [9] due to its
features like non-inflammatory, non-toxic and non-immunogenic properties [10,11]. So far,
several approaches have been developed for the preparation and processing of HAp including
electrodeposition [12], chemical precipitation [13], sol-gel method [14], flame synthesis [15],
microwave irradiation [16], solvothermal synthesis [17], hydrothermal synthesis [18],
neutralization method [19], Low- pressure synthesis [20], enzymatic hydrolysis [21],
precipitation method [22] and solid-state reaction method [23]. However, HAp from bio-wastes
such as fish scales [24], fish bones [25], eggshells [26], seashells [27] and bovine bones [10] are
still challenging in attaining nanostructured HAp.

Globally, more than 50% of fish capture is not produced as a valuable food product and
consists of million tons of unwanted bio-wastes [28]. Enormous amounts of fish by-products are
wasted and generating an undesirable environmental impact. Recently, fish scales waste has been
utilized as a low-cost source for the preparation of natural HAp. However, the properties of fish
waste synthesized HAp, and conventional commercial stoichiometric HAp is naturally distinct
from each other [21] and in its features. HAp prepared from fish waste revealed greater
biological activities due to the existence of essential anions like Cl- and F-, and cations like Mg2+,
Al3+, Sr2+, Zn2+, K+, and Na+, and the occurrence of both ions are showed better biomedical
applications mainly for the quick bone restoration [29]. Availability of Na+ and Mg2+ ions are
more critical for the growth and stability of bone and teeth, whereas their absence or the less
quantity may root the loss of bone or make it as fragile.

According to earlier reports, synthesize of HAp from fish scales is a simple and
economical method [21-24,30,31]. Kongsri et al. [30] reported the synthesis of HAp from

3
Tilapia nilotica fish scale waste and applied for the adsorption of selenium from aqueous
solutions. Similarly, Huang et al. [21] reported the preparation of HAp from Oreochromis
species (tilapia) fish scales using enzymatic hydrolysis method and examined the
biocompatibility. Abdulla et al. [31] reported the preparation of HAp from Black tilapia fish
scales using calcination method and confirmed the presence of Na+ and Mg2+ and Muhammad et
al. [24] reported the synthesis of HAp from Cyprinidae fish scales using ionic liquid
pretreatment and examined its biocompatibility. However, the entire above-mentioned methods
main drawback is hard to control the crystalline structure and size of HAp. Therefore, it’s
important to develop a simple, efficient green method to obtain biocompatible nanostructured
HAp for various biomedical applications.

In this work, we developed a simple alkaline heat treatment method for synthesizing of
nanostructured hydroxyapatite (NHAp) from Cirrhinus mrigala fish scale biowaste for the first
time. The surface morphology and chemical composition of the as-synthesized NHAp material
were characterized using various techniques including XRD, FT-IR, TGA, EDX and TEM. As-
synthesized NHAp material was utilized for further biocompatibility evaluation using MG-63
cells, and the results showed excellent biocompatibility which proven that Cirrhinus mrigala fish
scale biowaste produced NHAp could be a potential alternative bio-material for implantation
applications.

2. MATERIALS AND METHODS

2.1. Materials

MG-63 cells purchased from National Centre for Cell Science, Pune, India. Commercial
hydroxyapatite (CHAp) and p-Nitrophenyl phosphate bought from Sigma-Aldrich. Further,
Modified Eagle Medium (MEM), Giemsa stain, Fetal Bovine Serum (FBS), 1% penicillin, 1% L-
glutamine, 1% NEAA (Non-Essential Amino Acids), Sodium hydroxide pellet and Trypsin were
purchased from Hi-media. Double distilled and deionized water were used during the
experiments.

2.2. Collection of fish scale samples

4
 Cirrhinus mrigala fresh fish scales collected from the local fish market in Bhavani city,
Erode district, Tamil Nadu, India. Collected fish scales were washed using tap water followed by
double distilled water to remove dusty substances and air-dried at 40 °C for 3 hours and then
stored at room temperature for further studies.

2.3. Synthesis of NHAp

NHAp was synthesized from gathered fish scales by simple alkaline treatment. In brief,
500 mL of 0.1 M HCl was prepared and externally washed remove the protein from fish scales.
Afterwards, 250 mL of 5% (w/v) NaOH solution was added and heated at 70° C for 7 hours with
stirring (500 rpm) for the elimination of remaining proteins. After the 7 hours treatment, white
colour precipitates were obtained and washed three times using deionized water and kept
overnight in a hot air oven at 70° C. Subsequently, dried precipitates were carefully crushed as a
powder using mortar and pestle. Further, prepared powders were heated with 100 mL of 50%
(w/v) NaOH at 100° C and continuously stirred (500 rpm) for 2 hours. After the treatment
deionized water used to remove the alkaline nature of the synthesized NHAp and dried at 100 °C
[32].

2.4. Characterization of NHAp

The crystalline phase composition, diffraction lines of NHAp analyzed using XRD (Mini
flex II- X-ray diffractometer, Rigaku Co., Japan) with Cu-Kα radiation at 40 kV and 40 mA with
a 2Ɵ range of 0 to 80∘. Further, the Scherrer formula and Bragg reflection were used to calculate
crystalline size and lattice parameters, respectively. The purity and functional groups of NHAp
was measured using FT-IR (Bruker Tensor 27 spectrophotometer, Germany) in the range of
4,000 to 400 cm-1. Thermal stability of NHAp was examined using the simultaneous thermal
analyzer (PerkinElmer STA 6000, USA) at heating range of 30 to 900 ∘C. The structural
morphology of NHAp identified by Transmission Electron Microscopy (JEE 3010, JEOL, Japan)
and selected area electron diffraction (SEAD). Energy Dispersive X-Ray (EDX - Coupled with
SEM-Quanta-250-FEG, USA) performed to the analysis of the presence of elements.

2.5. Cell culture process

5
 The MG-63 cells were cultured in MEM added with 10% FBS, with adequate antibiotics
(Penicillin, Streptomycin and Gentamycin) in a culture flask, and it was maintained at 37∘C in a
humidified CO2 incubator with 5% CO2 supply. Sub-cultured was performed after cells reached
the 80% confluences and media was changed once in two days.

2.6. Cell viability study of NHAp

To estimate the cell viability of NHAp, MG-63 cells were used. The MTT (3-(4,5-
Dimethylthiazol-2-YL)-2,5-Diphenyltetrazolium Bromide) assay executed to measure the cell
viability and biocompatibility of NHAp. Concisely 1 x 104 cells were seeded into a 96 microwell
plate and permitted to adhere for 24 hours in CO2 incubator at 37∘C. After 24 hours, the new
medium was replaced, and the cells were treated with different concentrations of NHAp and
CHAp materials (5, 25, 50, 75 and 100 µg/mL) for 24 hours. Cells were gently washed with PBS
after treatment of NHAp and CHAp, then 20µl of MTT (5mg/mL) was added into each well and
incubates for 4 hours at 37∘C in a CO2 incubator. After incubation dissolves the formazan
crystals formation with 200µl of DMSO and mixed well. The color absorption of each sample
was measured by a multi-plate reader (Epoch, USA) at 570nm. The MG-63 cell without NHAp
was assigned as a control and assumes it is 100% cell viability. The results were expressed by %
of a number of viable cells.

2.7. Giemsa staining

For Giemsa staining, MG-63 cells separately treated with NHAp and CHAp samples
using 5, 25, 50, 75 and 100 µg/mL concentrations. Further, the cells allowed growing for 48
hours in the T-25 flask. After 48 hours, the medium was replaced and washed with PBS. The cell
fixation carried out using 50 % methanol for 3 min then washed with PBS. Next, to the fixation,
3 mL of 10 % Giemsa stain added in the flask for 10 min and washed with PBS. Finally, Stained
cells observed under Axiovert 25 inverted microscope (200µm) equipped with Zeiss camera.

2.8. Alkaline phosphatase activity of NHAp

Alkaline phosphatase (ALP) activity of MG-63 cells was examined after five days [21].
For ALP activity, samples washed with PBS then added to the lysed solution at 37∘ C for 30 min,
which contains 0.1 mM p-nitrophenyl phosphate and 1 M MgCl2. Afterwards, 1 M NaOH was

6
added to stop the activity of the enzyme. Further, Activity of ALP was measured using a multi-
plate reader (Epoch, USA) at 405 nm.

2.9. Statistical analysis

The MTT and ALP activity results designated as the mean ± standard deviation (SD) and
examined using ANOVA (one-way) Tukey’s HSD tests of variance, with a p-value of 0.05 being
significant, by the (IBM SPSS statistics 20) statistical software.

3. RESULTS AND DISCUSSION

3.1. XRD analysis of NHAp

The crystal structure and phase purity of the NHAp from Cirrhinus mrigala scales were
identified using XRD patterns (Fig. 1). All the observed NHAp peaks widely considered to
standards. Form, the XRD pattern of Cirrhinus mrigala fish scale NHAp, the observed diffracted
peaks well matched with the standard JCPDS 00-009-0432 (hydroxyapatite) which had the
(002), (102), (210), (211), (112), (300), (202), (301), (310), (311), (113), (203), (222), (312),
(213), (321), (402), (004), (214), (502), (304), (512), (432) and (513) planes. By comparing the
XRD results with JCPDS, confirms the formation of a perfect crystalline Hexagonal structure of
HAp [25, 30, 33]. The average ‘a’ and ‘c’ values of NHAp (a = 9.429 Å and c = 6.908 Å) almost
identical the lattice parameter of standard JCPDS 00-009-0432 (Hydroxyapatite). The XRD
based d-spacing values were measured and listed (Table S1). The unit cell parameters and degree
of crystallinity of NHAp depicted in Table 1. These results exhibit the NHAp has no impurity,
and the good crystalline structure found.

3.2. FT-IR analysis of NHAp

The FT-IR spectra of NHAp shown in (Fig. 2) wide bands at around 2400-3550 cm-1, and
1638 cm-1 are results of absorbed water. The small bands spotted at 3568 cm-1, and 629 cm-1 were
because of the hydroxyl group stretching vibrations. Peaks appeared at 575, and 608 cm-1
attributed to v4 bending mode of O-P-O group, 463 cm-1 to v2 symmetric mode as well as the
peaks at 1042 and 1094 cm-1 to v3 asymmetric stretching mode. The peak observed at 874 cm-1 is

7
resultant and confirmation of carbonate-based groups. The peaks at 1464 and 1416 cm-1
attributed to the v3 asymmetric stretching mode of carbonate and also evidenced that NHAp from
Cirrhinus mrigala is B type carbonate group [31, 34, 35]. Further, FT-IR spectrum shows the
presence of carbonate group in the NHAp synthesized from Cirrhinus mrigala scale wastes, as
recognized by characteristic peaks of carbonate at 1464 cm-1. The carbonate substitution is a
significant mineral state of the bone and has confirmed to have good osteointegration,
biocompatibility and bio-resorption equated to synthetic HAp [25, 36]. FT-IR spectra also reveal
there is no formation of impurities.

3.3. TGA analysis of NHAp

The TGA results displayed weight loss of NHAp in two steps within temperature range
from 30 to 900 ∘C (Fig. 3). In first step, weight loss take place (2 %) at 50 to 350 ∘C due to the
water loss in the NHAp. Further, broad peak occurred at 400 to 750 ∘C because of the
crystallization process [37]. The final and second weight loss (3 to 6 %) appeared at 800 to 900
∘C corresponds to the loss of hydroxyl groups and minor decomposition. The synthesized NHAp
TGA result showed a good thermal stability and low level of weight loss (6 %) compared to the
other reports [37, 38].

3.4. EDX analysis of NHAp

Fig. 4 represents the result of EDX analysis of NHAp. EDX analysis indicated the
existence of Ca, P, C, O, Na and Mg in NHAp. Generally, the presences of Na and Mg ions have
a significant factor in bone and teeth growth [39]. In other reports researchers prepared HAp with
different Ca/P molar ratios using fish scales and bones waste [33, 40, 41], which were near to
theoretical value of Ca/P Molar ratio level. However, our synthesized NHAp Ca/P molar ratio
value was approximately 1.67 (Table S2), which is almost the same and matched with the
theoretical Ca/P molar ratio (1.67) value of HAp.

3.5. TEM analysis of NHAp

The morphological appearance of the NHAp synthesized from Cirrhinus mrigala fish
scale was examined by transmission electron microscopy and was presented in Fig. 5a-b.
According to TEM analysis performed at different magnifications and it shows that Cirrhinus

8
mrigala NHAp are in the agglomerated rod-like nanostructured shapes particles with average
size of 30-50 nm [42-44]. From the SAED pattern (Fig. 5c) of the NHAp, the calculated d-
spacing values are well matched with the values calculated through XRD (Table 2).

3.6. Cell biocompatibility study of NHAp

The viability of NHAp and CHAp towards MG-63 cells analyzed, and the MTT assay
carried out after 48 hours indicated in Fig. 6. According to the MTT results, NHAp considerably
enhances the MG-63 cells viability at different dosages compared with CHAp (Table S3).
Further, the concentration variation of (5 to 100 µg/mL) NHAp displayed better cell viability
compared to the same concentration of CHAp (Fig. S1). In previous, Pal et al. [45] reported the
synthesis of HAp from Lates calcarifer fish bones and used for the biocompatibility in MG-63
cells that reported cell viability was higher on commercial HAp compared to fish bone-derived
HAp. Similarly, Paul et al. [46] studied the preparation of HAp from Catla catla fish scale and
stated that the synthetic HAp has more cell viability compared to fish scale-derived HAp using
same MG-63 cells. However, our NHAp displays enhanced cell viability compared to
commercial HAp due to the presence of trace elements such as Na, Mg and the substitution
carbonates which is supported by preceding studies [25, 39]. Although, this study exhibited
effectiveness of NHAp towards promoting the cell viability and biocompatibility functions
compared to other reports [45, 46, 47].

The inverted microscopy images of stained MG-63 cells after the treatment of NHAp and
CHAp samples at different concentrations (5, 25, 50, 75 and 100 µg/mL) shown in image Fig. 7.
The surface of the NHAp different concentrations covered with more MG-63 cells compared to
CHAp concentrations due to the development of the mineral matrix, which is a significant
indicator for osteoinduction process (Fig. 7a-b) [21].

For better bone replacement, a material should fulfil the criteria of total bone-forming
ability and cellular activities. Therefore, it is a necessity to find out these things in our NHAp,
and the increases of ALP (which is an early marker of osteoblast differentiation, estimation of
the function osteoblast) activity depends upon the proliferation of osteoblasts. The ALP activity
of the MG-63 cells on CHAp and NHAp substrates for the 50 and 100 µg/mL concentrations
shown in Fig. 8 and ALP activity of MG-63 cells on NHAp from Cirrhinus mrigala fish scale is

9
higher than on CHAp at the time of 5th day of cell culturing observation. In both concentrations,
NHAp shows better ALP activity than the CHAp. The development of differentiation and
proliferation of MG-63 cells resulted not only for NHAp meanwhile it depends on various
factors such as Mg2+, Na2+ and CO32- availability promotes the interaction in MG-63 cells and
further, Mg2+, Na2+, and CO32- ions encourages cell differentiation, proliferation, formation and
adhesion of mineralized tissues [48]. The ALP results exhibited that NHAp directly promotes the
differentiation and proliferation of MG-63 cells. The outcome of MTT and ALP indicated that
the NHAp creates an appropriate condition for cellular activities and it may be a promising and
significant material in bone and teeth related biomedical applications.

4. CONCLUSION

This study proves an alkaline heat treatment method for the better formation of rod-like
shaped NHAp from Cirrhinus mrigala fish scale. The physical characterization results showed
that NHAp was in hexagonal structure with well matched with hydroxyapatite JCPDS and
presence of B type carbonate group without formation of any impurities. Further, synthesized
NHAp was rod-like shape nanostructured particles with average size of 30-50 nm and thermally
stable up to 900 ∘C. The biocompatibility (MTT, ALP assay, and Giemsa staining) of NHAp
compared with CHAp, the NHAp have higher cell viability and biocompatibility due to the exact
Ca/P molar ratio (1.67) and the presence of trace elements such as Na and Mg. Based on the
above results and discussions, we have concluded that NHAp from Cirrhinus mrigala fish scales
mainly useful in biomedical sciences for the better replacement of bones.

Acknowledgments
The authors gratefully thank the Department of Microbial Biotechnology, Bharathiar

University for extending the wet lab facility. The authors also thankful for the Ministry of

Science and Technology-Taiwan (MOST105-2113-M-037-019-MY2), and Research Center for

Environmental Medicine, Kaohsiung Medical University (KMU)-Taiwan for research grant

supports.

10
Conflict of interest
The authors have declared no conflict of interest.

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Fig. 1 XRD pattern of synthesized NHAp compared with JCPDS (00-009-0432).

17
Fig. 2 FT-IR spectra of synthesized NHAp.

18
Fig. 3 TGA graph of synthesized NHAp.

19
Fig. 4 EDX image of synthesized NHAp.

20
Fig. 5 TEM image at different magnifications 50 nm (a) 20 nm (b) and SAED pattern (c) of
synthesized NHAp.

21
Fig. 6 Effects of NHAp and CHAp on MG-63 cell viability, as assessed by an MTT method after
48 hours. The sign * indicate significant variance compared with CHAp concentrations (p <
0.05).

22
(a)

(b)

Fig. 7 Giemsa stained inverted microscopy (Axiovert 25 - 200µm range) images of NHAp (a)
and CHAp (b) at the different concentrations on MG-63 after 48 hours.
23
Fig. 8 ALP activity of NHAp and CHAp at 5 days. The sign * indicate significant variance
compared with CHAp concentrations (p < 0.05).

24
Graphical Abstract

25
 Table 1. Unit cell parameters and degree of crystallinity of NHAp.

Unit cell parameters (Å) Crystallinity

a c (Xc)
9.429 6.908 0.7183

Table 2. d-spacing value comparison using XRD and TEM patterns of NHAp.

Plane XRD TEM

(indexed as in XRD) d-spacing d -spacing
(Å) (Å)
211 2.812 2.858
310 2.274 2.277
213 1.847 1.845
004 1.725 1.739
502 1.503 1.509

Highlights
 Low-cost and green process to the synthesis of NHAp from Cirrhinus mrigala fish scale
 The biophysical characterization results show that agglomerated rod-shaped nanoparticles
 NHAp from Cirrhinus mrigala fish scale shows better biocompatibility compared to
CHAp.
 The ALP results indicated that NHAp promotes cell differentiation and proliferation.

26

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