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Hydroxyapatite and Hydroxyapatite-Chitosan Composite from Crab Shell

Article  in  Journal of Biomaterials and Tissue Engineering · December 2013

DOI: 10.1166/jbt.2013.1126

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 Copyright © 2013 American Scientific Publishers Journal of
All rights reserved Biomaterials and Tissue Engineering
Printed in the United States of America Vol. 3, 653–657, 2013

Hydroxyapatite and Hydroxyapatite-Chitosan

Composite from Crab Shell
Biswadeep Chudhuri1 , D. Bhadra2 , S. Dash3 , G. Sardar4 ,
K. Pramanik1 , and B. K. Chaudhuri3
∗
1
Department of Biotechnology and Medical Engineering, NIT Rourkela, Odisha 769008, India
2
Department of Materials Science, Indian Association for the Cultivation of Science,
Kolkata 700032, India

RESEARCH ARTICLE
3
Department of Physics, NIT Rourkela, Odisha 769008, India
4
Department of Zoology, Baruipur College, W.B. 743610, India

Natural crab shells (CS) is an important biological source of calcium. Like eggshell, CS also con-
tains CaCO3 along with Chitin. CS becomes flexible when treated with dilute acetic acid and NaOH.
Ca10 (PO4
6 (HO)2 or hydroxyapatite (HAp) layer is found to form on the surface of this flexible sheet
by soaking it in K2 HPO4 (KHP) solution (0.25–0.3 mol%) for several days. HAp-Chitosan composite
is also formed by immersing CS powder (250–300 m) in KHP solution. Crab shell may be con-
sidered as an important biological source of HAp and the HAp-Chitosan composite which might be
used for making scaffolds for tissue engineering and other applications.
Keywords: Crab Shell, Hydroxyapatite, Biomaterials, Bioceramics, Scaffolds.
Delivered by Publishing Technology to: unknown
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Copyright: American Scientific Publishers

1. INTRODUCTION
Hydroxyapatite (HAp) or Ca10 (PO4
6 (OH)2 is an important
bioactive and biocompatible material.1 It has got enormous
applications in bone and teeth repairing.2–4 This is mainly
due to excellent biocompatibility and bone bonding ability
of HAp. HAp is also the major mineral phase of bone.5
Scaffolds for tissue engineering applications can also be
made from HAp-Chitosan (or other biodegradable poly-
mers like PEG, PVA etc.) composites.6–8 There are sev-
eral biological sources of calcium like eggshell,9 10 snails11
etc. from which HAp can be obtained. Among these bio-
logical sources, crab shell (CS) appears to be typically
important as it contains both Chitin and CaCO3 . Chitin
(Fig. 1) present in CS is an important biocompatible and
biodegradable biopolymer with structural similarity to that
of glycosaminoglycans.12 13 Ge et al.14 showed that CS
itself is an important biomaterial containing CaCO3 and
Chitosan. It was reported earlier that the eggshell contain-
ing CaCO3 could be easily converted to CaO by burning
at 850  C and then to HAp by soaking CaO in K2 HPO4
(KHP) solution (0.25–0.30 Mol%).10 So there is possi-
bility of forming HAp from the CS if similar process is
adopted.
∗
Author to whom correspondence should be addressed.
In this paper our plan is to report that pure HAp powder
is automatically formed on the surface of the processed
CS by soaking it in KHP solution. No HAp-Chitosan
composite was formed on the surface of CS. We have also
shown that HAp-Chitosan composite can be obtained from
CS powder soaked in the same KHP solution. Both HAp
and HAp-Chitosan composites are important for making
scaffolds for tissue engineering applications.
2. MATERIALS AND METHOD
Chemicals used for converting CS to HAp were K2 HPO4
(99.8% pure, Loba, India), NaOH (99.8% pure, Merck,
India) and acetic acid (99.5%, Finar Chemicals Ltd.,
India). Crab shells (Figs. 2(a), (b)) were collected from
Sundarbans Area, West Bengal, India. The upper surface
of CS is little more reddish than the inner one which is
due to different CaCO3 and Chitin concentrations of the
two surfaces.
2.1. Formation of HAp from CS
It has already been pointed out that HAp is obtained by
soaking CaO in KHP solution. To estimate the amount of
KHP to be added to a particular amount of CS, one has
to estimate the amount of CaO available per gm of CS.
For this, after washing with water and drying, the CSs are

J. Biomater. Tissue Eng. 2013, Vol. 3, No. 6 2157-9083/2013/3/653/005 doi:10.1166/jbt.2013.1126 653

 Hydroxyapatite and Hydroxyapatite-Chitosan Composite from Crab Shell Chudhuri et al.

Fig. 1. Molecular structure of Chitosan and Chitin.

heated around 350  C at the heating rate of 3  C/min and
they were kept at this temperature for two hours. During
this heating process, organic substances (Chitin, CO2 etc.)
were removed and the shells became gray to black. The
RESEARCH ARTICLE
∼ 9 for thirty days at constant temperature (37  C). Lay-
ers of white substance were found to form gradually on
the surface of the CS tape. This white powder (Fig. 2(d))
collected from the surface of the shells was washed with

heating temperature of the furnace was then increased at
 
the rate of 5 C/min up to 850 C and the sample was
retained at this temperature for three hrs. During this heat-
ing process, CaCO3 present in the CS is converted to white
CaO powder (CaCO3 heated in air at 850  C → CaO +
CO2 ). Almost 20 g of raw CS, after burning at 850  C,
produced around 2.5 g of CaO. From this amount of CaO,
we estimated the amount KHP in the solution to keep the
Ca/P ratio around 1.67 (required for HAp formation). Then
the corresponding equivalent amount of CS was soaked
in (0.25–0.30 mol%) solution of KHP for HAp formation.
Before soaking CS in KHP solution, Delivered by Publishing
the natural
CS is Technology
IP: 117.194.145.180
immersed first in low concentration On: Thu,As27mentioned
(1 mol%) acetic acid
Copyright: American Scientific Publishers
overnight which makes CS flexible and strong, The cut
pieces of such CS are shown in Figure 2(c). The flexi-
ble CS is again soaked in dilute NaOH solution (2 mol%)
for deproteination of the surface for three hrs and then
they were washed with distilled water and dried in air.
The flexible dried CS sheets thus obtained were soaked
in calculated KHP solution (0.25 mol%) with pH around
di-ionized water and alcohol and dried at 100  C for 48 h.
About 1.5 g of such powder was collected from 10 g of
CS sheets. It is to be noted that this white powder (HAp)
formation is only a partial as conversion of CaCO3 , present
in the CS sheets, to HAp as complete conversion might
need more time. Here only CaCO3 present on the surface
layers of CS sheets reacted with KHP solution. Moreover,
amount of HAp production also depends on the thickness
and size of the CS (i.e., age of the shell which becomes
thicker with age), soaking time and pH of the solution.
2.2. Formation
of HAp-Chitosan Composite
to: unknown
Nov 2014above,
04:53:54
in our process, CS sheet soaked in
KHP solution did not form HAp-Chitosan composite on
the CS surface. Only pure HAp layers were formed. How-
ever, the processed powder of CS soaked in KHP solu-
tion was found to form HAp-Chitosan composite from CS
powder using the following process.
The CS powder (200–250 micron grain size) is first
obtained by grinding the dry CS. Before grinding, clean
CS was heated in the oven around 100  C for half an hour
to remove moisture. The CS powder was then treated with
dilute calcium acetate (1 mol%) for one hr and then in
NaOH (5 mol%) solution for five hrs. After washing and
drying, this powder was soaked in K2 HPO4 for thirty days.
The beaker containing the solution with CS powder was
kept at 37  C. The solution was changed every week end.
Finally the soaked powder was washed several times with
di-ionized water and dried under vacuum. This powder is
found to contain HAp-Chitosan composite. Here also com-
posite formation was not complete. Inner part of the grain
remained unreacted.

Fig. 2. (a) Upper and (b) lower surfaces of a crab shell (CS). (c) Pro-
cessed flexible crab shell sheets and (d) white (HAp) powder produced
on these shell sheets after soaking it in K2 HPO4 solution for thirty day
at 37  C.
2.3. Characterization
The structure and morphology of the white powder from
the CS sheets and the soaked CS powder were character-
ized by X-ray Diffraction (XRD), field emission scanning
electron microscopy (FESEM Model JEM-2010, JEOL).
X-ray diffraction (XRD) patterns using a Philips Shif-
fert 3710 diffractometer with using CuK radiation source

654 J. Biomater. Tissue Eng. 3, 653–657, 2013

Chudhuri et al. Hydroxyapatite and Hydroxyapatite-Chitosan Composite from Crab Shell

( = 154 Å) were obtained at room temperature. The

data were analyzed in the 2 range from 10 to 80 .
Fourier-transform infrared (FTIR) spectra were recorded
on a Perkin–Elmer spectrum 100 FTIR spectrometer with
a 4 cm−1 resolution in the range of 400–4000 cm−1 .

3. RESULTS AND DISCUSSION

Deacetylation of Chitin present in CS occurred when
treated with concentrated NaOH with the formation of
Chitosan. Figures 3(a) and (b) showed the field emission
scanning electron micrograph (FESEM) pictures of white
powder (HAp) formed on the processed CS surfaces after
soaking in KHP solution.
Figure 4(a) presents XRD of the white HAp powder (as
shown in Fig. 1(d)) formed on the CS surface. Observed

RESEARCH ARTICLE
peaks confirmed the formation of HAp. The XRD of the
KHP solution soaked CS powder shown in Figure 4(b)
also indicated the formation of HAp. The energy dispersive
X-ray (EDX) spectra of the CaO obtained by burning CS
around 850  C and HAp produced from the soaked CS are
Fig. 4. XRD of the white powder (HAp) produced on the surface of
shown in Figure 5 indicating the presence of appropriate
processed crab shell sheets after soaking in K2 HPO4 solution for thirty
amounts of Ca, O and P in CaO and HAp. days. (b) XRD of the same crab shell powder after soaking in K2 HPO4
Figure 6(a) shows the FTIR spectra of the CS derived solution for thirty days.
white HAp powder. The band at 3402 cm−1 belongs to the
vibration of hydroxyl. The band at 1035 cm−1 is the char-
acteristic band of phosphate stretching vibration, while the
bands at 875 and 564 cm−1 are due Delivered by Publishing
to phosphate bending Technology to: unknown
IP: 117.194.145.180 On: Thu, 27 Nov 2014 04:53:54
Copyright: American Scientific Publishers

Fig. 3. (a), (b) White clusters of (HAp) powder produced on the surface Fig. 5. EDX spectra showing the presence of Ca and O in CaO obtained
of processed crab shell sheets by soaking in K2 HPO4 solution for thirty from burnt CS (a) and the presence of Ca, P and O in the obtained HAp
days. from the K2 HPO4 solution soaked CS.

J. Biomater. Tissue Eng. 3, 653–657, 2013 655

 Hydroxyapatite and Hydroxyapatite-Chitosan Composite from Crab Shell
Fig. 6. FTIR spectra of the white (HAp) powder (a) and the crab shell powder (b) after soaking in K2 HPO4 solution for thirty days.
RESEARCH ARTICLE
vibrations. Carbonate ions occupy two different sites in
carbonate apatite peaks in the region 1650–1300 cm−1 due
to vibrational mode of carbonate ions.13 15 In Chitosan-
HAp composite, the Chitosan bands should appear around
744, 1629, 597 cm−1 , corresponding to HAp.16 Such peaks
are not found from the HAp powder (Fig. 2(d)) formed on
the CS surface. FTIR results also showed that the chemical
groups present in the CS derive white HAp powder sam-
ples match well with those of standard HAp powder. Our
present finding contradicts the result of Ge et al.14 who
showed HAp-Chitosan composite Delivered by the
formation on Publishing
surface Technology
IP: 117.194.145.180
of CS sheet by soaking in ammonium On: Thu,of27
phosphate solution.
Our method of HAp preparation fromCopyright: American
CS is, however, dif- Scientific
ferent from those of Ge et al.
However, HAp–Chitosan like composite formation is
indicated from the analysis of XRD (Fig. 4(d)) and FTIR
(Fig. 6(b)) spectra of the KHP solution soaked CS pow-
der sample. In this case, the 3150–3437 cm−1 range
widened showing the unique form of N H bond. The
band at 1000–1100 cm−1 and 500–600 cm−1 corresponds
to different modes of the PO4 group in HAp.17 18 Chitin
amide group has the characteristic peaks around 1650 and
1560 cm−1 which are relatively weaker in the spectra.
The bands at 1415–1485 cm−1 and at 870 cm−1 are due
to carbon ions in apatite. The bands 1550–1700 cm−1
are attributed to the superposition of HAp OH group and
Chitosan amide I and amide II groups. The bands at
−1 −1
3600–3700 cm and 2800–2900 cm are assigned to the
hydroxyl group present in Chitosan. Analysis of the FTIR
spectra of the KHP of the soaked CS powder indicates that
there is strong interaction between the AZS amide and the
hydroxyl group which shifts the HAp peak positions in the
FTIR spectra of CS forming HAp-Chitosan composite.
4. CONCLUSION
Finally, it is concluded that HAp layer is first formed on
the processed CS sheet. CaCO3 present in CS is slowly
transformed to CaO and CO2 . CaO immediately reacts
with K2 HPO4 solution and produce HAp.10 Chitin present
656
Chudhuri et al.
in CS does not interact. HAp-Chitosan composite can be
obtained from the reaction of CS powder and KHP solu-
tion. Therefore, CS is an important source of HAp and
even HAp-Chitosan composite, which might be treated as
scaffold for in vitro cells growing. Though it not very
beneficial to produce HAp directly from CS, as it is a
long time process, but HAp formation from the burnt CS
like burnt eggshell10 might be beneficial one. Moreover,
for making HAp-Chitosan composite, CS powder can be
used. Further research on the crab shells processing tech-
nique withto: theunknown
variations of different parameters like pH
theNov 2014 04:53:54
solution, temperature, soaking time etc. might be
Publishers
interesting. Artificially made micro porous CS sheet can
be made and used as scaffold for tissue engineering and
other applications.
Acknowledgments: One of the authors (B. K.
Chaudhuri) is grateful to CSIR, Government of India for
partial financial support. Authors The authors (Biswadeep
Chudhuri and K. Pramanik) acknowledge the financial
support provided by the Department of Biotechnology,
Government of India.
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Received: 25 June 2013. Accepted: 14 August 2013.

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