Materials Science and Engineering C 62 (2016) 183–189

Contents lists available at ScienceDirect

Materials Science and Engineering C

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

Hydroxyapatite from fish scale for potential use as bone scaffold or

regenerative material
Weeraphat Pon-On a,⁎, Panan Suntornsaratoon b, Narattaphol Charoenphandhu b,c, Jirawan Thongbunchoo b,
Nateetip Krishnamra b,c, I. Ming Tang d
a
Department of Physics, Faculty of Science, Kasetsart University, Bangkok, Thailand
b
Center of Calcium and Bone Research, Faculty of Science, Mahidol University, Bangkok, Thailand
c
Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand
d
Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand

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

Article history: The present paper studies the physico-chemical, bioactivity and biological properties of hydroxyapatite (HA)
Received 17 November 2015 which is derived from fish scale (FS) (FSHA) and compares them with those of synthesized HA (sHA) obtained
Received in revised form 18 January 2016 by co-precipitation from chemical solution as a standard. The analysis shows that the FSHA is composed of
Accepted 20 January 2016
flat-plate nanocrystal with a narrow width size of about 15–20 nm and having a range of 100 nm in length
Available online 22 January 2016
and that the calcium phosphate ratio (Ca/P) is 2.01 (Ca-rich CaP). Whereas, synthesized HA consists of
Keywords:
sub-micron HA particle having a Ca/P ratio of 1.65. Bioactivity test shows that the FSHA forms more new apatite
Hydroxyapatite than does the sHA after being incubated in simulated body fluid (SBF) for 7 days. Moreover, the biocompatibility
Fish scale study shows a higher osteoblast like cell adhesion on the FSHA surface than on the sHA substrate after 3 days of
Bone tissue engineering culturing. Our results also show the shape of the osteoblast cells on the FSHA changes from being a rounded
Natural resource shape to being a flattened shape reflecting its spreading behavior on this surface. MTT assay and ALP analysis
Bone scaffolds show significant increases in the proliferation and activity of osteoblasts over the FSHA scaffold after 5 days of
culturing as compared to those covering the sHA substrates. These results confirm that the bio-materials derived
from fish scale (FSHA) are biologically better than the chemically synthesized HA and have the potential for use
as a bone scaffold or as regenerative materials.
© 2016 Published by Elsevier B.V.

1. Introduction is different from the natural hydroxyapatite in human bone in terms

Trauma, certain diseases, and physical force may lead to fracture
of bone and may result in dangerous complications, especially in the
elderly. In recent years, biomaterials based on calcium phosphate
especially hydroxyapatite (HA) have been widely used to repair bone
fractures and other defects because of their osteoinductive and
osteoconductive properties [1–4]. Other features which make hydroxy-
apatite attractive for use as bone replacement are that it is the
major component of bones and hard tissues, is non-toxic and non-
immunogenic, has the mechanical strength and has the surface proper-
ties desirable for bone tissue regeneration [2]. For these reasons, it has
been widely used in orthopedics and dental applications [3]. Most of
the studies on the use of HA as bone substituted materials employ
chemically synthesized hydroxyapatite (sHA) (from solutions containing
calcium and phosphate ions) and the nanostructures with different
morphologies including nanorods, nanobundles and nanoparticles have
been prepared via a simple precipitation method [5–9]. However, sHA
⁎ Corresponding author.
E-mail address: fsciwpp@ku.ac.th (W. Pon-On).
of several of their physicochemical properties such as their strength
and chemical composition [10,11], which leads to sHA having lower
biological activity than natural hydroxyapatite. Based on the premise
that if the properties (such as chemical composition and structure) of
biological HA are preserved at the nanoscale level, beneficial effects
due to the HA properties could be achieved and the biomaterial would
be better accepted by the living organs (and be biologically safe). The
stratagem of extracting them from natural sources has been previously
investigated. In recent year, various natural materials, such as fish
bones, fish scale and mineral materials have been used to produce HA
[12–20]. Chemical analysis reveals that these products are abundant
sources of calcium phosphate and could be used as an economic source
for HA synthesis. In addition, these materials would be seen as being
by-products (biowaste) from the food industry. The conversion of such
biowaste would be considered to be one of the benefits for a solid
waste management, reduced environmental impact.
To study the natural derived HA products for use as bone substituted
materials, Milovac et al. and Beom-Su et al. [12,13] used cuttlefish-bone-
derived hydroxyapatite (CB-HA). They compared the properties of the
CB-HA derived materials with those of the synthetic hydroxyapatite.

http://dx.doi.org/10.1016/j.msec.2016.01.051
0928-4931/© 2016 Published by Elsevier B.V.
184 W. Pon-On et al. / Materials Science and Engineering C 62 (2016) 183–189
Their results showed that CB-HA was more non-toxic and exhibited
greater cell adhesion, proliferation and differentiation when compared
to those of synthetic HA. HA derived from the bones of sword fish
(Xiphias gladius) and tuna (Thunnus thynnus) revealed that these mate-
rials were non-toxic and could be a promising new bioactive material
[14]. Research on producing HA from waste shells were investigated
by Shavandi et al. [15] and Tâmâsan et al. [16]. Based on their results,
it was proposed that these HAs had the potential to be materials for
use in bone tissue engineering applications. Fish scales are another
source of natural materials which could be used to synthesize HA. The
preparation of HA from fresh water fish scale (Labeo rohita) and bio-
compatibility was done by Modal et al. [19]. They found that the cells
grown on the scaffold made with these types of HA materials promoted
cellular attachment and proliferation. However, the conversion of such
raw materials into HA, an alkaline heat treatment method (thermal
treatment at 800 °C with basic CaCl2) had to be employed to obtain
the HA ceramics. Such a thermal treatment led to a HA/β-TCP biphasic
material and could change the physicochemical and biological activities
of the original materials. In this paper, we obtained the HA material
from the alkaline hydrolysis fresh Probarbus jullieni scales (FS) without
the high temperature treatment. These FSHAs were characterized by
several analytical techniques to determine their composition and
microstructural features and tested for their biological activity. These
properties were then compared to the properties of the synthetic HA
grown from solution.
2. Experimental
2.1. Materials
Chemicals used in this experiment are Ca(NO3)2·4H2O (calcium
nitrate) (Fluka Chemika, Switzerland), (NH4)2HPO4 (diammonium
hydrogen phosphate) (Fisher Scientific, UK), sodium hydroxide
(NaOH) (Merck, Germany), NH4OH (Merck, Germany) and hydrochloric
acid (HCl) (Mallinckrodt chemicals, USA). They were used without
further purification.
2.2. Extraction of hydroxyapatite from fish scale (FSHA)
The scales were taken from the fresh water fish (P. jullieni) raised in a
fish farm located in Ratchaburi Province, Thailand. The details of the
preparations being; the fresh P. jullieni scales (FS) were first rinsed
thoroughly with water to remove any grease. The adhering tissue was
manually scraped. Thereafter, the scales were soaked and stirred in 4%
hydrochloric acid (HCl) solution for 15 min at room temperature for
deproteinization. Afterwards the solution was neutralized by sodium
hydroxide (NaOH) treatment to obtain hydroxyapatite-rich slurry and
was subsequently filtered through a filtered-pressed machine having a
500 μm diameter pore membrane. The resulting obtained FSHA cake
was sealed in a plastic bag and boiled in distilled water at 100 °C
for 30 min to deactivate the enzymes, and kept at − 20 °C for
further used. Before characterization, the FSHA was dried at 60 °C in a
hot air oven.
2.3. Synthesis of hydroxyapatite (sHA)
Synthesis of calcium phosphate of hydroxyapatite (sHA)
powder was done by an aqueous precipitation reaction. Briefly,
Ca(NO 3 )2 ·4H2 O (0.40 M) and (NH4 ) 2HPO4 (0.24 M) solutions
were gradually mixed together at room temperature, with the simulta-
neous adjustment of pH solution to 11 with NH4OH. The supernatant
solutions were then removed from the powder. The resulting powders
were washed with de-ionized water until the pH was reduced
to about 7 and then freeze-dried. Before performing the physico-
chemical and biological measurements, samples of the powders were
heated to 500 °C for 2 h in an ambient air atmosphere to thoroughly
dry them.
2.4. Characterization of FSHA and sHA
The crystal structures of the composite pellets were determined
by powder X-ray diffraction (XRD) (Bruker diffractometer, Model D8
Advance) using the CuKα radiation and operating at 40 kV and 40 mA
current. The angles scanned were from 2θ = 10° to 60° at a scanning
speed of 1 incremental step of 0.037° per second. For the FT-IR absorp-
tion measurements, the powders were mixed with KBr and pressed
into pellets using a pressure of 8 tons for 1 min. The pellets were ana-
lyzed using a Fourier transform infrared (FTIR) spectrophotometer
(Spectrum GX, PerkinElmer) which performed 16 scans over the
range 370–4000 cm−1. A scanning electron microscope (SEM) (JEOL
model JSM-6301F) was used to observe the changes in size and
morphology of the samples. An accelerating voltage of 15 kV was used
to obtain the SEM images. The nanocrystals of particles were also
examined by a transmission electron microscope (TEM) (JEOL model
JEM-2010). For the mechanical testing, the loads for the compres-
sive testing of the FSHA and sHA were dried cylindrical scaffolds
of 7 mm in diameter and an average height of around 13 mm
(height/diameter ≈ 2) were tested with a crosshead speed of
5 mm/min using a mechanical tester (Universal Testing Machine
(Instron Model 55R4502, S/NH3342)).
2.5. In vitro biomineralization of sHA and FSHA scaffolds
To study the bioactive behaviors of sHA and FSHA scaffolds, each
of the prepared powders was pressed in pellets (having diameters
of ~1.3 cm and ~2.5 mm in thickness) and then soaked in a simulated
body fluid (SBF) for 7 days. The SBF solution we used is one of the
more extensively used ones (see ref. [21]). It contains the following
chemicals: NaCl (136.8 mM), NaHCO3 (4.2 mM), KCl (3.0 mM),
K2HPO4 (1.0 mM), MgCl2·6H2O (1.5 mM), CaCl2 (2.5 mM) and Na2SO4
(0.5 mM) and is buffered at pH 7.4 with tris(hydroxymethyl)
aminomethane [(CH2OH)3CNH2] and hydrochloric acid (HCl). The
bioactivity of each of the sHA and FSHA samples was assessed by
immersing the pellets in 50 mL of SBF for one week at 37 °C. The SBF
is replaced every three days to avoid any changes in the cationic concen-
tration that may occur due to degradation of the sample. After immer-
sion in the SBF, the pellet sample was washed with de-ionized water
before the SEM analysis.
2.6. Attachment and morphology of cell on glass, FSHA and sHA substrates
(biological test)
For the biological test, as previously described in Ref. [21], rat
osteoblast-like UMR-106 cells (derived from Sprague–Dawley rats
which are rapidly growing osteoblast-like cells. They usually become
confluent within 3–4 days in 6-well plate or culture disc) [American
Type Culture Collection (ATCC) No. CRL-1661] were grown in a
Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO,
USA) supplemented with 10% fetal bovine serum (FBS; PAA, Pasching,
Austria) and 100 U/mL penicillin–streptomycin (Gibco, Grand Island,
NY, USA). The cells were propagated in a 75-cm2 T-flask (Corning, NY,
USA) in a humidified atmosphere containing 5% CO2 at 37 °C. The
media also contained a pH indicator (phenol red), which becomes
yellow in acidic pH and was sub-cultured as described in the ATCC
protocol. Confluent UMR-106 cells used for the experiment were
obtained by seeding cells at 1 × 106 cells/Petri dish into 100-mm petri
dish in which circular glass (diameter ~ 1.3 cm), FSHA and sHA discs
(diameter ~1.3 cm) had been placed. The Petri dishes were incubated
in a humidified atmosphere containing 5% CO2 at 37 °C. The culture
medium was replaced every 3 days. At days 3, 5 and 7 after seeding,
the glass, FSHA and sHA discs were examined for cell attachment and
 W. Pon-On et al. / Materials Science and Engineering C 62 (2016) 183–189
morphology by SEM. The glass, FSHA and sHA discs were first briefly
rinsed in cold 0.1 mol/L phosphate buffer (pH 7.2) twice. The cells
were then fixed in 2.5% glutaraldehyde (Electron Microscopy Science,
Fort Washington, PA, USA) in 0.1 mol/L phosphate buffers for 3 h. The
fixative was removed and cells were rinsed in 0.1 mol/L phosphate
buffer and in distilled water. Thereafter, the glass, FSHA and sHA discs
were dehydrated in a graded series of ethanol solution (50%, 70%,
80%, 90% and absolute ethanol). The glass, FSHA and sHA discs were
desiccated under a vacuum and were coated with copper before the
cell morphology, cell adhesion and cell proliferation were to be studied
by means of SEM.
2.7. Analysis of viability and osteogenic differentiation of rat osteoblast-like
UMR-106 cells cultured onto the substrates
Cell viability and osteogenic differentiation of rat osteoblast-like
UMR-106 cells cultivated in the scaffolds were determined by measur-
ing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) activity and the alkaline phosphatase (ALP) activity, respectively.
Regarding the MTT assay, the amount of formazan produced is
proportional to the number of viable cells present. This method is also
described in ref. [21]. The details are: The cells were first incubated
on the scaffolds being studied for 3, 5, and 7 days. After being
trypsinization, harvest suspension was centrifuged and re-suspended
in 1000 μL of media. A hundred microliter of suspension was transferred
to a 96-well plate and incubated with 10 μL (0.5 mg/mL) MTT solution
for 3.5 h in the dark. Upon centrifugation, the supernatant was removed,
and replaced with 150 μL of MTT solvent (4 mM HCl, 0.1% Nonidet P-40
(NP40) in isopropanol) to dissolve the purple formazan crystals.
Fig. 1. SEM and EDS analyses of FSHA (a, c) and sHA powders (b, d), respectively.
185
The absorbance of the solutions at 595 nm was then measured using a
micro-plate reader (Model M965, Metertech Inc., Taipei, Taiwan).
To measure the ALP activity, the lysates were incubated with
1 mg/mL p-nitro-phenylphosphate (Sigma) in 0.1 mol/L diethanolamine
(pH 9.8) containing 1% Triton X-100 and 1 mmol/L MgCl2 at 37 °C for
30 min. NaOH (1 mol/L) was then added to stop the enzymatic reaction.
After centrifugation at 16,000 g for 10 min, the supernatant was trans-
ferred to a microtiter plate and its absorbance was determined at
405 nm (Model M965, Metertech Inc., Taipei, Taiwan). This process
was measured in duplicate. Cell viability and ALP activity were shown
as percentage of cell grown on glass. Data was analyzed with the
unpaired Student's t-test (FSHA vs sHA; n = 4–5 each). Statistical
significance was considered to be P b 0.05.
3. Results and discussion
3.1. Physico-chemical characterization of synthesized FSHA and sHA
powders
The SEM images of fish scale-derived HA and sHA are shown in
Fig. 1. The analysis shows that the FSHA powders (Fig. 1(a) and figure
inset) are composed of flat-plate like morphology of calcium phosphate
in the form of cells of submicron average size. The synthetic hydroxyap-
atite exhibits a regular microscale spherical morphology. The FSHA has a
porous structure, large surface area and higher surface roughness,
all of which differs from dense and agglomerated uniform structure
of sHA (Fig. 1(b) and figure inset). Previous studies indicate that the
cell adhesion correlates with substrate surface roughness [22–24],
thus suggesting that FSHA can be used for bone tissue engineering
186 W. Pon-On et al. / Materials Science and Engineering C 62 (2016) 183–189
application. Due to its having a porous structure, the compressive
strength of FSHA will be slightly lower compared to sHA, i.e., up to
7.41 MPa and 8.38 MPa for FSHA and sHA, respectively (data not
shown). The strength of FSHA is in good agreement with the cancellous
human bone (i.e., 2–20 MPa) [25].
Results of elemental composition together with Ca/P ratio of the
powders obtained are shown in Fig. 1(c and d). The main components
of the powders obtained from both the fish scale extraction and the
synthetic hydroxyapatite are calcium and phosphorous elements in
the samples with the average Ca/P weight ratio of around 2.01 and
1.65 for FSHA and sHA, respectively. These are slightly higher (for
FSHA (Ca-rich HA)) and lower (for sHA (Ca-poor HA)) than the
stoichiometric ratio of 1.67 for apatite in human bone [4–11]. The higher
Ca/P ratio obtained in FSHA substrates compared to apatite can be
attributed to the presence of carbonate ions substituted phosphate in
the apatite structure (B-type carbonate hydroxyapatite). The presence
of B-types of carbonate hydroxyapatite in our fish scale extraction and
synthetic hydroxyapatite is confirmed by the FT-IR spectra (Fig. 2).
The peak at 875 cm− 1 can be assigned to the bending mode B-type
carbonate hydroxyapatite (lower intensity for sHA) [26]. The peaks at
1460 and 1419 cm−1 are associated with bending in B type carbonate
hydroxyapatite due to the substitution of PO34 − by CO23 −. The FT-IR
shows similar spectra in both samples at around 3000–3700 cm− 1
which are characteristic of the –OH stretching of H2O. The second region
contains the peaks at 1093–1047 cm− 1 which are due to the PO34 −
stretching mode. In the band between 579 and 607 cm− 1 are the
characteristic peaks of PO3−4 bending mode [14,15,19,26].
The XRD pattern of the FSHA and sHA is shown in Fig. 3. The sample
consisted entirely of HA phase with well-defined peaks given in the
JCPDS card No. 72-1243 of HA. As seen from Fig. 3, the XRD peaks of
sHA are higher and sharper when compared with those of FSHA
powders, thus indicating that better crystallization of synthetic HA
particles had occurred. The broad peak from XRD diffraction data can
be used to estimate the crystal size in a direction perpendicular to the
crystallographic plane based on Scherer's formula, d = kλ / βcosθ,
where d is the average crystallite size, λ is the wavelength of X-ray
radiation (0.154056 nm), and k is a constant related to the crystal
shape and is approximately equal to unity. β is the full width of the
peak at half of the maximum intensity (rad) and θ is the Bragg's angle.
The average size of apatite crystal is calculated using the (002)
reflection peak (2θ = 26°). The sample shows a nanosized crystal of
23.11 and 40.37 nm for FSHA and sHA, respectively.
The TEM images of nanocrystals of FSHA and sHA are shown in Fig. 4.
It is seen that the crystal of FSHA is a mix phase of rod-shaped crystal
and a flat-plate nanocrystal. This further confirms the morphology
Fig. 2. The FT-IR spectra of FSHA and sHA powders.
Fig. 3. XRD pattern of hydroxyapatite from FSHA powders.
exhibited by the SEM image (Fig. 4(a)). From TEM results, the narrowest
size plates are about 15–20 nm in width and around 100 nm in length
while the rod like nanocrystals are of about 50 nm in diameter and
lower than 100–200 nm in length for FSHA and sHA, respectively. The
nanostructure of FSHA determined from TEM and XRD studies agrees
well with the average crystallite size in natural bone. These crystallites
were probably formed by fusion of fundamental blocks [26,27].
Moreover, it has been reported by previous researchers that this
rod-like shape typical for stoichiometric apatite can be attributed to
the substituent elements such as the coupled CO−3 −4
2 to PO3 (confirmed
by FTIR as seen in Fig. 2) which in turn cause changes in the size and
shape of the apatite crystal [14]. Generally, these bone-substituting
materials have as good bioactivity and osteoconductivity as those
observed in HA composed of nanosized crystals [2,4].
3.2. In vitro bioactivity of sHA and FSHA scaffolds
Fig. 5 (a-b) shows the morphologies of sHA and FSHA scaffolds after
immersion in the SBF solution for 7 days. The surfaces show apatite
particles forming on them but with a higher apatite particle density
on the FSHA scaffolds with the particle sizes being around 5 μm in
diameter. This difference in behavior can be accounted for by assuming
that the FSHA uses the Ca-rich HA on their surfaces to interact with the
phosphate ion in the fluid and this interaction (resulting in changes in
the surface structure) is the formation of the apatite. sHA (the Ca-poor
HA) on the other hand appears to crystallize into apatite gradually as
the surfaces of the sHA stabilize in SBF. The aggregations of calcium
phosphate of apatite species on the surfaces of the scaffolds were con-
firmed by EDS. EDS (Fig. 5(c, d)) measurements confirmed the presence
of Ca and P on the surface of these particles. The EDS spectrum indicated
that the ratio of Ca/P was 1.66 and 1.65 for sHA and FSHA, respectively.
These are close to the Ca/P ratio of the hydroxyapatite in natural bone
(Ca/P is 1.67) [4–11]. The results from the SEM/EDS data confirm that
the FSHA has a superior ability to induce apatite formation. This is of im-
portance since the ability of a material to form apatite on its surface
upon SBF soaking would be crucial if one is interested in the formation
of interfacial bonds between the apatite and bone when one is using
FSHA as an in vivo implant material.
3.3. Biological properties of synthesized FSHA and sHA powders
(in vitro test)
The cell adhesion and cell viability on the materials must be
measured in order to determine whether the synthesized materials
 W. Pon-On et al. / Materials Science and Engineering C 62 (2016) 183–189 187

Fig. 4. TEM images of hydroxyapatite nanocrystals of FSHA (a) and sHA (b).

have the potential to be used as scaffolds for bone regeneration. Fig. 6 extended length as compared with the cell grown on sHA. The results
shows the morphologies of rat osteoblast-like UMR-106 cells grown of the cell viability (Fig. 7(a)) studies indicated that cell viabilities on
on the scaffolds after 3, 5 and 7 days of culture. The SEM images revealed hydroxyapatite derived from fish scale are higher to those formed on
that the cells cultured on the FSHA substrates show more flatness and sHA after 7 days of culturing. The viabilities of the cells grown on both

Fig. 5. SEM/EDS images of FSHA (a, c) and sHA (b, d) scaffolds after 7 days in SBF, respectively.
188 W. Pon-On et al. / Materials Science and Engineering C 62 (2016) 183–189

Fig. 6. SEM images of osteoblast cells on glass (positive control), FSHA and sHA scaffolds after 3, 5 and 7 days of culture, respectively.

substrates are decreased on the 5th day of culturing. This could be due
to changes in the physical properties such as the density, porosity or
crystallinity of the scaffolding surface which would result in unsuitable
bone-cell ingrowth. In light of this report agreement with previous
report, biological HA showed better adherence and stimulated prolifer-
ation of osteoblast cells than sHA [13,18].
Determination of the overall cellular activity and the bone-
formation ability is important to the evaluation of FSHA as bone replace-
ment materials. We have therefore carried out an alkaline phosphate
activity (ALP) assay, an early marker of osteoblast differentiation, to
measure the osteoblast function. Fig. 7(b) shows the proliferation and
ALP activity of the UMR-cells cultured on FSHA and sHA substrates
with respect to glass. We find that ALP activity of the cells on FSHA is
higher than on sHA during the period between the 3rd day and 7th
day of culturing. The ALP activity of the cells on the HA-derived from
fish scale increased markedly as the culture time is increased. This
demonstrates that there is a high degree of FSHA material–osteoblast
interaction when compared to that with the sHA substrate. The osteo-
blast proliferation and differentiation results cannot be explained as
being due solely to the types of calcium phosphate used since they de-
pend on many factors. Previous studies have shown that the physical
structure such as the crystalline formation or surface roughness includ-
ing surface area leads to increase cell adhesion and proliferation. In ad-
dition, the HA-derived from natural materials is expected to contain
essential mineral ions that lead to enhanced cell adhesion, proliferation,
differentiation and formation of mineralized tissue [13,14]. Although we
did not directly evaluate the effect of these substituent elements on
osteoblast differentiation, we measured ALP activity because it is one
of the most commonly used markers for the determination of osteoblast
differentiation. When UMR-106 cells were cultured with substrates, the

Fig. 7. Cell viability on FSHA and sHA scaffolds as determined by MTT assay (a) and the relative alkaline phosphatase (ALP) activities (b) of osteoblast-like UMR106 cells after 3, 5 and 7 days
of culture (glass substrate as positive control paired with each material). * b 0.05; *** b 0.05 compared with the corresponding control group (mean ± SE).
 W. Pon-On et al. / Materials Science and Engineering C 62 (2016) 183–189
ALP activity was higher on FSHA than on pure sHA (Fig. 7(b)). This
observation suggested that the FSHA provided a suitable environment
for cellular activities.
4. Conclusions
In this study, the physicochemical and biological performance of HA
derived from P. jullieni scale is investigated. The results are compared to
those when synthetic HA is used. The characterizations reveal that the
FSHAs are in the form of flat-plate nanocrystal and have a porous
morphology, large surface area and higher surface roughness which
lead to increase cell adhesion and proliferation when compared to
sHA. The studied SBF confirmed that the FSHA has a superior ability to
induce apatite formation. The present data shows that the osteoblastic
cells prefer to adhere to the surface of the FSHA better than to the sHA
substrate. Based on quantitative cellular measurements (MTT assay
and ALP activity) the FSHA has much better biochemical properties.
The combination of large surface area, higher surface roughness,
increase cell adhesion and proliferation properties point to the FSHA
having the better potential for application in bone tissue engineering.
Acknowledgments
The authors would like to acknowledge the financial support from
the Kasetsart University Research and Development Institute
(KURDI43.59), Thailand Research Fund (TRF)—Kasetsart University
(TRG5780269, to Pon-On W.), and the Department of Physics, Faculty
of Science, Kasetsart University. We thank Mr. Thitiwat Siripaskongpat
and team for P. jullieni scale preparation. This work is supported by
Mahidol University (to Charoenphandhu N.) and Thailand Research
Fund (TRF)-Mahidol University (DPG5780004 to Krishnamra N. and
RTA5780001 to Charoenphandhu N.). Thongbunchoo J. was supported
by Faculty of Science, Mahidol University.
References
[1] A. Sugawara, K. Asaoka, D. Shinn-Jyh, Calcium phosphate-based cements: clinical
needs and recent progress, J. Mater. Chem. B. 1 (2013) 1081–1089.
[2] S. Samavedi, A.R. Whittington, A.S. Goldstein, Calcium phosphate ceramics in bone
tissue engineering: a review of properties and their influence on cell behavior,
Acta Biomater. 9 (2013) 8037–8045.
[3] A.J. Salinas, M. Vallet-Regí, Bioactive ceramics: from bone grafts to tissue engineering,
RSC Adv. 3 (2013) 11116–11131.
[4] P. Wang, L. Zhao, J. Liu, M.D. Weir, X. Zhou, H.H. Xu, Bone tissue engineering via
nanostructured calcium phosphate biomaterials and stem cells, Bone Res. 2
(2014) 14017.
[5] M. Sadat-Shojai, K. Mohammad-Taghi, E. Dinpanah-Khoshdargi, A. Jamshidi,
Synthesis methods for nanosized hydroxyapatite with diverse structures, Acta
Biomater. 9 (2013) 7591–7621.
[6] C. Combes, C. Rey, Amorphous calcium phosphates: synthesis, properties and uses in
biomaterials, Acta Biomater. 6 (2010) 3362–3378.
189
[7] F. Mohandes, M. Salavati-Niasari, Simple morphology-controlled fabrication of
hydroxyapatite nanostructures with the aid of new organic modifiers, Chem. Eng.
J. 252 (2014) 173–184.
[8] F. Mohandes, M. Salavati-Niasari, Particle size and shape modification of hydroxyap-
atite nanostructures synthesized via a complexing agent-assisted route, Mater. Sci.
Eng. C 40 (2014) 288–298.
[9] F. Mohandes, M. Salavati-Niasari, Influence of morphology on the in vitro bioactivity
of hydroxyapatite nanostructures prepared by precipitation method, New J. Chem.
38 (2014) 4501–4509.
[10] S.V. Dorozhkin, M. Epple, Biological and medical significance of calcium phosphates,
Angew. Chem. Int. Ed. 41 (2002) 3130–3146.
[11] J. Zhang, W. Liu, V. Schnitzler, F. Tancret, B. Jean-Michel, Calcium phosphate cements
for bone substitution: chemistry, handling and mechanical properties, Acta
Biomater. 10 (2014) 1035–1049.
[12] D. Milovac, G.G. Ferrer, M. Ivankovic, H. Ivankovic, PCL-coated hydroxyapatite
scaffold derived from cuttlefish bone: morphology, mechanical properties and
bioactivity, Mater. Sci. Eng. C 34 (2014) 437–445.
[13] K. Beom-Su, H.J. Kang, Y. Sun-Sik, L. Jun, Comparison of in vitro and in vivo
bioactivity: cuttlefish-bone-derived hydroxyapatite and synthetic hydroxyapatite
granules as a bone graft substitute, Biomed. Mater. 9 (2014) 025004.
[14] M. Boutinguiza, J. Pou, R. Comesaña, F. Lusquiños, A. de Carlos, B. León, Biological
hydroxyapatite obtained from fish bones, Mater. Sci. Eng. C 32 (2012) 478–486.
[15] A. Shavandi, A. El-Din, A. Bekhit, A. Ali, Z. Sun, Synthesis of nano-hydroxyapatite
(nHA) from waste mussel shells using a rapid microwave method, Mater. Chem.
Phys. 149-150 (2015) 607–616.
[16] M. Tâmâsan, L.S. Ozyegin, F.N. Oktar, V. Simon, Characterization of
calcium phosphate powders originating from Phyllacanthus imperialis and
Trochidae Infundibulum concavus marine shells, Mater. Sci. Eng. C 33
(2013) 2569–2577.
[17] N.N. Panda, K. Pramanik, L.B. Sukla, Extraction and characterization of biocompatible
hydroxyapatite from fresh water fish scales for tissue engineering scaffold,
Bioprocess Biosyst. Eng. 37 (2014) 433–440.
[18] H. Yi-Cheng, H. Pei-Chi, C. Huey-Jine, Hydroxyapatite extracted from fish scale:
effects on MG63 osteoblast-like cells, Ceram. Int. 37 (2011) 1825–1831.
[19] S. Mondal, A. Mondal, N. Mandal, B. Mondal, S.S. Mukhopadhyay, A. Dey, S. Singh,
Physico-chemical characterization and biological response of Labeo rohita-derived
hydroxyapatite scaffold, Bioprocess Biosyst. Eng. 37 (2014) 1233–1240.
[20] A.S. Kamba, Z.A.B. Zakaria, Osteoblasts growth behaviour on bio-based calcium
carbonate aragonite nanocrystal, Biomed. Res. Int. (2014).
[21] W. Pon-On, N. Charoenphandhu, J. Teerapornpuntakit, J. Thongbunchoo, N.
Krishnamra, I.M. Tang, Mechanical properties, biological activity and protein
controlled release by poly(vinyl alcohol)–bioglass/chitosan–collagen compos-
ite scaffolds: a bone tissue engineering applications, Mater. Sci. Eng. C 38
(2014) 63–72.
[22] R.A. Gittens, R. Olivares-Navarrete, Z. Schwartz, B.D. Boyan, Implant osseointegration
and the role of microroughness and nanostructures: lessons for spine implants, Acta
Biomater. 10 (2014) 3363–3371.
[23] H. Ivankovic, G.G. Ferrer, E. Tkalcec, S. Orlic, M. Ivankovic, Preparation of highly
porous hydroxyapatite from cuttlefish bone, J. Mater. Sci. Mater. Med. 20 (2009)
1039–1046.
[24] K. Beom-Su, K. Jin Seong, S. Hark-Mo, Y. Hyung-Keun, L. Jun, Cellular attachment and
osteoblast differentiation of mesenchymal stem cells on natural cuttlefish bone,
J. Biomed. Mater. Res. A. 100A (2012) 1673–1679.
[25] S. Bose, M. Roy, A. Bandyopadhyay, Recent advances in bone tissue engineering
scaffolds, Trends Biotechnol. 30 (10) (2012) 546–554.
[26] P.N. Kumta, C. Sfeir, L. Dong-Hyun, D. Olton, D. Choi, Nanostructured calcium
phosphates for biomedical applications: novel synthesis and characterization, Acta
Biomater. 1 (2005) 65–83.
[27] L.C. Palmer, C.J. Newcomb, S.R. Kaltz, E.D. Spoerke, S.I. Stupp, Biomimetic systems
for hydroxyapatite mineralization inspired by bone and enamel, Chem. Rev. 108
(2008) 4754–4783.