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Materials Today: Proceedings 4 (2017) 11933–11938 www.materialstoday.com/proceedings

ICNANO 2016

Low Temperature Development of Nano-Hydroxyapatite from

Austromegabalanus psittacus, Star fish and Sea urchin
Komalakrishna Ha, Shine Jyoth T.Ga, Biswanath Kundub, Saumen Mandala*
a
Department of Metallurgical and Materials Engineering, National Institute of Technology Karnataka (NITK), Surathkal-575025, India.
b
Bioceramics and Coating Division, CSIR-Central Glass and Ceramic Research Institute, Kolkata-700032, India.

Abstract

The study focuses the preparation and characterization (physico-chemical and mechanical) of hydroxyapatite [Ca10(PO4)6(OH)2]
(HAp) from naturally occurring dead marine species widely available in Arabian sea shore; namely Austromegabalanus
psittacus, star fish (Asteroidea) and sea urchin (Echinoidea). All three marine species were found to be source of calcium
carbonate in the form of aragonite (calcite) that crystallize in an organic matrix. The calcined aragonite was converted to nano-
sized hydroxyapatite powder by chemical reaction with Ortho-phosphoric acid while maintaining stoichiometry, Ca/P = 1.67 at
80 oC. It was found from XRD that the powders were composed of pure HAp with average crystallite size of 10 nm. SEM, on
the other hand, revealed nano-rod like elongated structures having a length in the range of 100 to 700 nm with an aspect ratio of
3.5. Further, the HAp powders were used to prepare cylindrical pellet samples by uni-axial pressing, sintered at 1100 oC
temperature, made flat-parallel, polished and used finally for assessment of elastic modulus by compression test method.
Primary results showed typical elastic modulus of pellets from Austromegabalanus psittacus is 3 GPa, star fish 2.1 GPa and sea
urchin 2.3 GPa (L/D = 1.3 and strain rate = 0.01 mm/s). Thus, the powder synthesized from marine source could be a potential
alternative for development of structural bio-ceramics and also can be used as scaffolds for bone or dental implants, because of
its easy and economical fabrication.

© 2017 Elsevier Ltd. All rights reserved.

Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON NANOTECHNOLOGY (ICNANO-
2016).

Keywords: Marine species, aragonite, nano-hydroxyapatite, chemical synthesis, low temperature

*
Corresponding author. Tel.: +91-0824-2473752
E-mail address: [email protected]

2214-7853© 2017 Elsevier Ltd. All rights reserved.

Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON NANOTECHNOLOGY (ICNANO-2016).
11934 Komalakrishna H. et al / Materials Today: Proceedings 4 (2017) 11933–11938

1. Introduction

Nano-hydroxyapatite (HAp); [Ca10 (PO4)6(OH)2] is an active biological material can be develop from marine
species, applicable in bone implantation and tissue engineering [1]. HAp, synthesized artificially can be used to
repair of damaged bone or teeth, due to its similarity of chemical composition and crystallographic structure to the
bio mineral in the natural tissue. HAp participate in the metabolic process of an organism, this is designed to
degrade gradually and replaced by the host tissue [2]. Calcium phosphate biomaterial exhibits wide range
application; could be useful such as bone defect filling, bone reconstruction, bone replacement drug carrier, coatings
on metal prostheses and even nerveguide [3]. Abundant availability of marine species leads to produce the HAp. A
number of investigations include shells of some marine mollusks such as a sea coral, nacre, sea snails and cuttlefish
bones etc, are the prominent examples. HAp synthesis from Tropical sea snail shells (Tiger Cowrie–Crypraea
Tigris) was reported by Sahin et.al.using chemical synthesis method [4]. Agaogullari et.al. produced biocermic
from sea urchin by ultrasonic and hotplate method [2]. Natural gypsum, natural calcite, bovine bone and cuttlefish
shell used to produce corresponding HAp [5]. Malaysian cockle shell CaCO3 is a bio ceramic is potential for
multiple engineering applications [6]. HAp synthesis from dead snail shells has reported with excellent properties
so essential for hard tissue replacement [7]. Biphasic bioceramic nano-powders HAp from shell of a regular sea
snail from Cerithiumvulgatum used as bioceramic graft material [8].
Although various chemical techniques are used to produce HAp, few noble methods are namely, precipitation
method, microwave processing, hydrothermal techniques, wet chemical precipitation and hydrolysis of calcium
phosphates [9]. HAp synthesized from marine species is more economical compared to commercially available
HAp, thanks to low cost chemical synthesis. Natural sources have the advantages that they inherit some properties
of raw material [10]. An increasing number of compounds are currently being isolated from aquatic organisms and
these have application in bioactive ingredients to medical devices [11].
In this work, nano hydroxyapatite was synthesized at low temperature from naturally occurring three abandoned
marine species namely Austromegabalanus psittacus, sea urchin (Echinoidea) and star fish (Asteroidea) (Fig.1)
through chemical synthesis route, which was investigated through thermal, crystal, morphological and spectroscopic
analysis. To obtained elastic modulus, cylindrical pellet was made which was sintered at elevated temperature.

Fig.1. Optical image of dead (a) Austromegabalanus psittacus, (b) Sea urchin, (c) Star fish.

2. Experimental procedure

Dead Austromegabalanus psittacus, Sea urchin and Star fish was collected from sea shore of Arabian Sea. After
cleaning of the species with hot water, it was crushed with agate motor to produce fine powder of Aragonite
(CaCO3). Then calcination of aragonite was carried out to at 900 °C to obtain CaO. Calcined powder (CaO) was
dissolved in distilled water and Ortho-phosphoric acid (H3PO4) was added drop-wise in such a manner as to
 Komalakrishna H. et al / Materials Today: Proceedings 4 (2017) 11933–11938 11935

maintain the stoichiometric molar ratio of Ca/P equal to 1.667 with continuous stirring using hot plate with magnetic
stirrer with a 200 rpm. Hot plate kept at 80 ºC for 2 hours for a successful completion of reaction; than the final
product was kept in hot air oven at100º C for overnight to remove the moisture content [4, 10, 12]. The same
procedure has been followed for all three kind marine species. The flow chart of process and chemical reaction
involved is shown in Fig.2. Pellets with a length/ diameter (L/D) ratio of 1.3, starting from HAp powders, were
prepared using Uni-axial hydraulic press with die and plunger, followed by sintering at 1100 ° C for two hours in the
furnace.

Fig.2. Flow chart of process and chemical reaction involved in the synthesis process of Hydroxyapatite.

Raw form of powders and synthesised HAp powders are characterized using different techniques. The thermal
analysis was determined using differential scanning calorimeter (DSC, NETZSCH DSC 404 F1). Crystalline phases
present in powders were studied by X–ray diffractometer (XRD, RIGAKU Miniflex 600). Morphological analysis
was carried out by scanning electron microscopy (SEM, JEOL model JSM 6380 system). The Fourier transform
infrared (FTIR) spectra of the produced HAp powders were analysed with JASCO FTIR -4200, JAPAN.
Cylindrical pellets were prepared from all three marine species from HAp powders using Uni-axial hydraulic
pressing (AIE UTM- 40T) to study the mechanical properties of bulk sintered HAp. Elastic modulus was calculated
through compression test using universal testing machine (UTM, SHIMADZU AG- X plus 100 KN).

3. Results and Discussion

3.1 Characterization of Aragonite

X-ray diffraction of three marine species is shown in Figure 3, where all peaks matched with aragonite, CaCO3
(according to JCPDS-000050586). This highly crystalline aragonite was the staring materials, subjected to be
analyzed with thermal study to understand the calcination temperature. Differential scanning calorimetry was
shown in Figure 3b and 3c. Major conversion temperature, CaCO3 to CaO, was obtained in DSC, which are
783.30C, 859.60C and 7450C for Austromegabalanus psittacus, sea urchin and star fish accordingly. Curve clearly
shows the decomposition of aragonite to CaO. Along with this decomposition peak, there is an another exothermic
peak at 5730C in sea urchin and star fish is due to the presence of silica; which is intense in star fish (Fig 3c).

Fig.3. (a)XRD plot of CaCO3(Aragonite), (b) DSC analysis of Sea urchin, (c) DSC analysis of Star fish raw powders.
11936 Komalakrishna H. et al / Materials Today: Proceedings 4 (2017) 11933–11938

3.2 Characterization of CaO (Calcined aragonite)

All three types of aragonite powder was calacined at 900oC for two hours, which is well above to the conversion
temperature (CaCO3 to CaO; was identified in the DSC analysis. Calcined CaO was characterised by XRD analysis
showing the Crystallite phases according to the JCPDS-371497, is shown in Fig.4 (a).Peaks at 2θ of 20.8° (100), 26°
(101), 45.8° (201) and 64° (113) correspond to silica (JCPDS-00-033-1161), is observed in case of starfish and sea
urchin.

3.3Characterization of HAp powder

The phases of HAp (JCPDS-740565) were obtained from XRD analysis of prepared HAp powder is shown in
Fig.4. (b). There is a clear indication of formation of HAp in all three cases. The peak width is the effect of
nanocrystallanity of HAp powders. The crystallite size is calculated using Debye-Scherrer’s method D = Kλ/β cos θ
where K is a dimensionless shape factor (0.9), λ is the x-ray wavelength, β is the line broadening width at half
maximum intensity (FWHM), θ is the diffraction angle and D is the crystallite size. Average crystallite size is
estimated to be 10 nm.

Fig.4.XRD analysis of (a) calcined aragonite(CaO), (b)synthesized HAp powders from three marine species.(c) FTIR spectra for HAp powders.

Fig. 4. (c). Displays the FT-IR spectra HAp powders synthesized from three marine species, is obtained by
potassium bromide (KBr) pellets method with an average 32 scans in the range of wavenumber of 4000 to 400 cm-1.
The results show the characteristic absorption peaks of the sample HAp nanoparticles [13]. The broad bands at
3424.1 and 2426.1 cm-1was attributed to adsorbed water, the peak at 3775 cm-1 was due to the stretching vibration of
the lattice OH-ions. The characteristic bands for PO4- appear at 569, 885.16, 1066.66 and 1421 cm -1. Our FTIR
result is exactly similar to previously reported result [14].

3.4 Morphological study of HAp powder

The morphology of HAp powder prepared by three marine species was studied by scanning electron microscope
(SEM) shown in fig. 5. Images indicate that powders are apparently rod like or needle shaped particles. Average
particle size (length) as calculated from SEM micrographs was found to be in the range of 100 to 700 nm. Average
size 597 nm in Austromegabalanus psittacus, 270 nm in sea urchin and 370 nm in star fish. Austromegabalanus
psittacus HAp structure looks like number of elongated irregular shaped rods. Sea urchin based HAp structures have
bigger agglomerates than other two, and Star fish having Small irregular rods [4]. Rod-like crystals are unstable as
compare to cube like crystals which makes them preferable for bone regeneration [5].
 Komalakrishna H. et al / Materials Today: Proceedings 4 (2017) 11933–11938 11937

Fig.5. SEM images of synthesized HAp powder from (a) Austromegabalanus psittacus, (b) sea urchin and (c) star fish.

3.5 Mechanical analysis of HAp pellets

Cylindrical pellets with length/diameter (L/D) ratio of 1.3 from HAp powders of three marine species were
prepared with the help of Uni-axial hydraulic pressing machine. Prepared pellets were sintered at relatively lower
temperature 1100 °C for two hours compared to others report [7]. Using UTM machine load applied in the rate 0.01
mm/second. Plot of stress vs strain was obtained & plotted as shown in fig. 6. Ultimate compression strength of
three cases was reported to be 75 N/mm2, 54 N/mm2 and 46 N/mm2 in case of Austromegabalanus psittacus, sea
urchin and star fish respectively. Elastic modulus was found to be 3 GPa, 2.3 GPa and 2.1 GPa in three cases, which
is lesser value. This is mainly due to low sintering temperature (1100 °C). Presence of silica in the star fish could
be one of the reasons for poor mechanical response. Although sea urchin contains trace amount of silica, compare
to star fish, but effect is nullified due to better packing in the green compact due to powder morphology.

Fig.6. Stress -Strain curve of HAp pellets from (a) Austromegabalanus psittacus, (b) sea urchin and (c) star fish.

4. Conclusions

Nano-Hydroxyapatite from three different marine species was synthesized successfully using Novel Chemical
method. XRD and FTIR characterizations clearly reveal the formation of Hydroxyapatite. Presence of silica
reduced value of young’s modulus of HAp’s. Higher sintering temperature is required to obtained higher young’s
modulus value of sintered powder. Pellet obtained from Austromegabalanus psittacus, star fish, sea urchin
exhibits Young’s modulus of 3 GPa, 2.1 GPa and 2.3 GPa, respectively; although there was a presence of silica; but
powder morphology plays an important role to retain mechanical properties. The production and characterization of
bio-ceramic powders of natural biologic origin from the three kinds of marine species was achieved and this can be
considered as promising materials for bone fillers.
11938 Komalakrishna H. et al / Materials Today: Proceedings 4 (2017) 11933–11938

Acknowledgements

The authors would like to thank Dr. Udaya Bhat K., Dr. Anandhan Srinivasan and Dr. Ravishankar K. S.,
Department of Metallurgical and Materials Engineering, National Institute of Technology Karnataka, Surathkal,
India, for providing laboratory facilities.

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