J Mater Sci: Mater Med (2007) 18:1735–1743

DOI 10.1007/s10856-007-3069-7

A novel route for synthesis of nanocrystalline hydroxyapatite from

eggshell waste
D. Siva Rama Krishna Æ A. Siddharthan Æ
S. K. Seshadri Æ T. S. Sampath Kumar

Received: 6 December 2005 / Accepted: 31 May 2006 / Published online: 5 May 2007
Ó Springer Science+Business Media, LLC 2007

Abstract The eggshell waste has been value engineered stability in phosphate buffer and cell culture test using
to a nanocrystalline hydroxyapatite (HA) by microwave osteoblast cells establishes biocompatibility of OHA.
processing. To highlight the advantages of eggshell as
calcium precursor in the synthesis of HA (OHA), synthetic
calcium hydroxide was also used to form HA (SHA)
following similar procedure and were compared with a Introduction
commercially available pure HA (CHA). All the HAs were
characterized by X-ray powder diffraction (XRD) method, Natural biomaterials like bone substitutes from bovine
Fourier transform infrared (FT-IR) spectroscopy, scanning source, cardiovascular prostheses of biological origin,
electron microscopy (SEM), transmission electron wound dressings made of biologically derived calcium
microscopy (TEM) and specific surface area measure- alginate, collagen, chitin etc., have recently started to
ments. Nanocrystalline nature of OHA is revealed through surpass the synthetic biomaterials by providing an abun-
characteristic broad peaks in XRD patterns, platelets of dant source for novel biomedical applications [1].
length 33–50 nm and width 8–14 nm in TEM micrograph Hydroxyapatite (HA), the inorganic constituent of bone
and size calculations from specific surface area measure- and hard tissues, is one of the most widely used biomate-
ments. FT-IR spectra showed characteristic bands of HA rials for reconstruction of the skeleton. Commercially
and additionally peaks of carbonate ions. The cell available HAs are expensive due to the use of high purity
parameter calculations suggest the formation of carbon- reagents [2, 3]. The HA derived from natural materials
ated HA of B-type. The OHA exhibits superior sinterabil- such as bovine bone [3–5], fish bone [6] or coral [7, 8] has
ity in terms of hardness and density than both SHA the advantage that they inherit some properties of the raw
and CHA may be due to larger surface area of its spheru- materials such as the pore structure, carbonated HA etc.
lite structure. The in vitro dissolution study shows longer However, problems do arise due to the variability of
physical and chemical properties of the raw material [3].
The eggshell represents 11% of the total weight of the egg
and is composed predominantly of calcium carbonate
(94%), calcium phosphate (1%), organic matter (4%) and
magnesium carbonate (1%). Though these are occasionally
D. Siva Rama Krishna
A. Siddharthan
used as a fertilizer due to their high content of calcium and
S. K. Seshadri
T. S. Sampath Kumar (&)
nitrogen [9], most of these are discarded as waste. By
Department of Metallurgical & Materials Engineering, Indian
Institute of Technology Madras, Chennai 600 036, India utilizing this organic waste, namely eggshell, the cost of
e-mail: [email protected] high quality calcium source for preparation of HA can be
avoided and at the same time recycling of eggshell also can
Present Address:
be taken care of [9]. Eggshell after treating with dilute
D. Siva Rama Krishna
School of Materials Engineering, Nanyang Technological sodium hypochlorite has been studied for application in
University, Singapore 639798, Singapore facial reconstructive surgery [2]. However calcium

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1736 J Mater Sci: Mater Med (2007) 18:1735–1743

carbonate is unsuitable for most implant purposes due to its and convert the calcium carbonate to calcium oxide which
high dissolution rate and poor stability [1]. HA materials in turn on exposure to atmosphere forms calcium hydrox-
which were prepared from bovine and human resources by ide. The product was finely ground in an agate pestle and
treating them with dilute HCI acid is not safe as some mortar. The calcium hydroxide was weighed and mixed
diseases can survive all controlled processes. For example with distilled water to form 0.3 M suspension and reacted
prions, a small proteinaceous infectious particle, can sur- with 0.5 M diammonium hydrogen phosphate solution
vive such processes. High temperature calcinations above corresponding to the stoichiometric ratio of Ca/P = 1.67.
850 °C could solve this problem of transmission of dis- The mixed reactants were irradiated in a domestic micro-
eases [10]. HA has been produced from eggshell at ele- wave oven (BPL India, 2.45 GHz, 800 W power). The
vated temperature of 1,050 °C, but contained minute microwave power and duration of exposure were optimized
fractions of other calcium compounds [9]. Highly sinter- to prevent spilling out of the container. The product was
able b-tricalcium phosphate (b-TCP) has been synthesized then washed repeatedly with distilled water to remove
from eggshell by a wet chemical method followed by unwanted ions and dried overnight in an oven at 100 °C.
heating at high temperature and the ball-milled b-TCP was This method of OHA synthesis from eggshell has been
fully densified at 1,200 °C [11]. repeated several times and found to be reproducible. The
Natural bone minerals are nanostructed non-stoichiom- SHA was prepared in an identical manner by microwave
etric HA of dimensions 20 nm in diameter and 50 nm long processing using synthetic calcium hydroxide (Analytical
with substitution of ions like magnesium, fluoride and grade, Merck, Germany) while the high purity CHA was
carbonate. From bionics viewpoint, synthetic apatites to be procured commercially (Aldrich, USA). A small amount of
used for repairing damaged hard tissues are expected to all the samples were heated at 900 °C for 2 h and furnace
have characteristics closer to those of biological apatite in cooled to improve the crystallinity and to check the purity.
both composition and structure [12]. Nanocrystalline HA The X-ray powder diffraction (XRD) analysis of the
has been shown to exhibit enhanced bioactivity [13]. samples was done (Shimadzu, XD-D1, Japan) in reflection
Microwave synthesis is an efficient and simple method for mode with Cu Ka radiation. The functional groups present in
preparing nanocrystalline inorganic materials with narrow HA were ascertained by Fourier transform infrared spec-
distribution of particle size and shape in a rapid manner troscopy (FT-IR). The FT-IR spectra were obtained over the
[14]. In recent years, there have been many reports of region 400–4,000 cm–1 using KBr pellet technique with
microwave synthesis of HA and influence of process spectral resolution of 4 cm–1. The crystalline size and mor-
parameters on its thermal stability, shape and size [15–21]. phology were analysed in a transmission electron microscope
Production of calcium carbonate and biphasic calcium (TEM) operated at 120 KeV (Philips CM12wSTEM,
phosphate materials by microwave processing of natural Netherlands). TEM specimens were prepared by depositing a
aragonite and calcite has also been reported [22]. few drops of HA dispersed in ethanol on carbon coated
In this study (based on which a patent has been applied copper grid. The specific surface area of the HA powder were
in India), a novel yet simple method to convert hen’s measured using surface area analyzer (CE instruments,
eggshell into nanocrystalline HA by microwave processing Sorpomatic, India) based on Brunauer Emmett and Teller
has been presented for the first time to the best of the (BET) gas adsorption method. The simultaneous thermo
knowledge of the authors. The physico-chemical charac- gravimetric analysis (TG) and differential thermal analysis
terizations of the nano HA powder were carried out. Since (DTA) of the organic waste was performed (Netzsch STA
HA can also been used in its ceramic form, fabricated by 409 thermal analyzer, Germany) in nitrogen atmosphere from
sintering [23], sinterability studies of the synthesized nano room temperature to 1,400 °C at a heating rate of 10 °C/min.
HA have been carried out. Dissolution rate and in vitro cell Sintering studies were carried out on samples of HA
culture studies were undertaken. To highlight the advan- pellets (10 mm diameter and 3 mm height) compacted at a
tages of using eggshell as a calcium source for the syn- pressure of 80 MPa, at various temperatures from 800 to
thesis of HA (OHA), the results were compared with HA 1,250 °C for 12 h in air. The sintered pellets were sub-
(SHA) prepared using synthetic calcium hydroxide by jected to XRD, hardness and density measurements.
similar microwave processing as well as with a commer- Density was determined using Archimedes principle with
cially available high purity HA (CHA). water as liquid medium using the formula: q2 = A/P
q0
(g/m3), where q2 is the density of the pellet, q0 is the
density of the liquid, A is weight of the pellet and P is the
Materials and methods difference in weights of the pellet before and after
immersion in the liquid. The hardness values were mea-
Hen’s eggshells were washed thoroughly and heated in a sured using a micro hardness tester (Shimadzu N 0 4800,
box furnace at 900 °C for 2 h to decompose organic matter Japan) at a load of 300 g applied for 10 s on the finely

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J Mater Sci: Mater Med (2007) 18:1735–1743 1737

polished surface of the sintered pellets. The microhardness of calcium carbonate to calcium oxide. This calcium oxide
was calculated from the indentations using the formula: on exposure to atmosphere would convert to calcium
Hv = 1.854 L/d2, where Hv is the Vickers hardness num- hydroxide. The XRD pattern of powdered eggshell
ber, L is the applied load in kg and d is the diagonal length (Fig. 2a) shows peaks corresponding to calcium carbonate
of the square indentation in mm. The morphology of HA (JCPDS 5-0586) only while the heat-treated eggshell
powder and fractured surface of sintered HA were studied (Fig. 2b) shows peaks corresponding to calcium hydroxide
using scanning electron microscope (JEOL JSM 5410 & (JCPDS 4-0733). Trace amounts of carbonate peaks
JSM 5300, Japan) after coating with a gold film. observed might be due to reaction with atmosphere to form
The dissolution study of HA powder was carried out in calcium carbonate while being furnace cooled as was
phosphate buffer solution (at a ratio of 1 mg/ml) of pH 7.2 observed in the heat treatment of corals [8].
maintained at 37 °C by measuring the pH at regular inter- The XRD pattern of oven dried OHA was found to be
vals for 1 week. The in vitro cytotoxicity test was done as similar to that of oven dried SHA and as received CHA,
per direct contact method (ISO 10993–5, 1999) using exhibiting peaks corresponding to HA (JCPDS 9-432) only
osteoblast cells maintained in minimum essential media while the results of heat-treated samples of OHA and SHA
supplemented with foetal bovine serum. These cells were show increased crystallinity with sharper peaks. Appear-
seeded onto 900 °C sintered HA pellets of 4 mm diameter ance of new peaks corresponding to b-TCP (JCPDS 9-169)
and 2 mm thickness for 24 h and viewed under optical was observed in case of CHA as shown in Fig. 3. This may
microscope. High-density polyethylene and copper were be due to its non-stoichiometric composition resulting in
used as negative and positive control samples respectively. decomposition at 900 °C [24]. The lattice parameters cal-
culated from XRD data by least squares fit method as listed
in Table 1, indicates that the values are comparable with
Results and discussion reported values of a-axis 9.418 Å and c-axis 6.884 Å
(JCPDS 9-432). The broad peaks observed in Fig. 3 indi-
Eggshell has a three layer structure consisting of outermost cate nanocrystalline nature of the synthesized powder. The
protein rich cuticle layer, the spongeous middle layer and average crystallite size of the powder was estimated using
the inner lamellar layer. Both the middle and inner layers the Scherrer formula t = K k/B Cosh, where t is the aver-
form a matrix constituted by protein fibres bonded to cal- age crystallite size (nm); K is the shape factor (K = 0.9); k
cium carbonate (calcite) crystals in the proportion of 1:50 is the wavelength of the X-rays (k = 1.54056 Å for Cu Ka
[9]. So, to evaluate the decomposition behaviour of the radiation); B is the full width at half maximum (radian) and
organic matter and calcium carbonate, the thermal analysis h is the Bragg’s diffraction angle (°). The diffraction peak
of eggshell was carried out. The simultaneous TG-DTA at 25.9° (2h) was chosen for calculation of the crystallite
thermogram of eggshell (Fig. 1) shows a weight loss of size, as it is isolated from others. This peak corresponds
45% between 50 and 830 °C and a sharp endothermic peak to (002) Miller plane family. The (002) peaks of oven
at 830 °C in DTA, which may be due to the decomposition dried OHA and SHA are broader than CHA indicating

250
(101 )

120
exo

(001)

1
200
(102)

100
* calcium carbonate
r e l a ti v e i n t e n s i t y ( a . u )

(100)

(110)
% Weight loss

(111)

0
DTA (µV/mg)

*
(201)

150 (b)
(112)

(211)
(202)
(003)

(210)

(203)
(002)

* *
*
80
(104)

100
-1

60
50
(113)

(01 8 )
(116)

(2 1 10)
(006)

(202)

(0 0 12)

(0 2 10)
(012)

(1 10 )

(a)
(211)

(300)
(122)
(214)

(128)

(226)

-2
(125)

(134)

0
40 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
200 400 600 800 1000 1200
2 theta (deg)
Temperature(oC)
Fig. 2 XRD pattern of powdered eggshell (a) and after heating at
Fig. 1 TG-DTA thermogram of eggshell 900 °C (b)

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1738 J Mater Sci: Mater Med (2007) 18:1735–1743

that of HA. The bands at 3,435 and 1,637 cm–1 correspond

(211)
β -- β -TCP to adsorbed H2O while bands at 1,482 and 1,424 cm–1 are

(112)
(300)

(213)
that of CO23 ions which suggests incorporation of car-
(002)

(310)

(222)
(102)

(301)
(210)

(004)
(312)

(321)
(410)
(402)
(200)
(111)

(212)
(f) bonate ion in synthesized HA [26]. The cell parameter of
OHA shows decreased a-axis characteristic of B-type
carbonate ion substitution [27].
(e)
Intensity (arb. unit)

β
The SEM micrographs of OHA, SHA and CHA powder
β β β
are shown in Fig. 5. From the SEM pictures, it is clearly
β β
(d) observed that the precipitated crystallites of both the OHA
and SHA were of spherulite morphology with narrow size
distribution of about few micron in diameter, whereas CHA
has flake like morphology of relatively larger size. The
(c)
spherulite is composed of tiny nanosize platelets of loosely
aggregated stabilized structure [18]. Hence, to characterize
(b)
the morphology of the platelets, TEM analysis was done on
ultrasonically dispersed OHA in ethanol. The bright field
(a)
TEM image of oven dried OHA powder is shown in Fig. 6.
The morphology clearly indicates that the particles are
20 30 40 50 60
platelets, mostly agglomerated and of dimensions: length
2 theta (degree)
33–50 nm and width 8–14 nm. This result thus clearly
Fig. 3 XRD pattern of as received CHA (a), oven dried SHA (b), confirms the nanosize nature of OHA.
oven dried OHA (c) and 900 °C heated CHA (d), SHA (e) and OHA The specific surface area of the OHA, SHA and CHA
(f) powdered samples obtained from the BET method is listed in Table 1. It is
observed that both the microwave processed HA’s had
nanocrystalline nature of microwave synthesized HAs. The larger specific surface area than the commercially pure HA
crystallite sizes as listed in Table 1, also confirm the and can be attributed to their particle size and shape
nanocrystalline nature of OHA. The fraction of crystalline (Fig. 5). As the nano particle size decreases, the surface
phase (Xc) of HA was evaluated using the following for- area increases which may be due to increased number of
mula [25], Xc = 1 – (V112/300/I300), where I300 is the atoms occupying the surface. The specific surface area of
intensity of (300) diffraction peak and V112/300 is the OHA (106 m2/g) has been found to be greater than reported
intensity of the hollow between (112) and (300) diffraction value of around 34–80 m2/g for nanocrystalline HA
peaks of HA. The Xc of OHA, SHA and CHA are listed in [28, 29]. The particle size was also calculated from BET
Table 1. The calculation shows that the crystalline fraction specific surface area using the empirical equation t = 6/
of OHA is slightly higher than SHA and CHA samples. (q
S) (where t is the average grain size in micron, q the
This may be due to the biological origin of calcium car- density in g/cm3 and S the specific surface area in m2/g)
bonate used. The heat treated OHA and SHA has Xc of 0.91 [28]. The values as listed in Table 1, again corroborates
and 0.9 respectively. XRD and TEM results characterizing nano dimension of
The FT-IR spectra of oven dried OHA, SHA and as microwave synthesized HAs. The SEM picture of CHA
received CHA samples are shown in Fig. 4. All the sam- shows the presence of flakes of micron size but calculations
ples exhibits characteristic stretching and librational modes based on the XRD results and surface area measurements
of OH– groups at 3,571 cm–1 (mS) and 631 cm–1 (mL), (Table 1) indicate nano size particles.
whereas the internal modes corresponding to the PO34 The in vitro dissolution study as shown in Fig. 7 indi-
groups occur at: 1,088 and 1,046 cm–1 (m3), 962 cm–1 (m1), cates that all three HAs are stable in phosphate buffer
599 and 560 cm–1 (m4) and 469 cm–1 (m2) corresponding to solution at pH 7.2, except for the initial fluctuations. The

Table 1 List of cell parameter, crystallite size, fraction of crystalline phase (Xc), specific surface area, density and particle size of HAs
Sample a(Å) c(Å) Crystallite Fraction of crystalline Surface area Density Particle size (nm)
size (nm) phase (Xc) (m2/g) (g/cm3) based on BET

OHA 9.412 6.877 21 0.13 106 3.12 18

SHA 9.410 6.872 19 0.09 104 3.06 19
CHA 9.439 6.88 29 0.11 76 3.04 26

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J Mater Sci: Mater Med (2007) 18:1735–1743 1739

OHA
that the Ca/P ratio of SHA may be less than the stoichi-
875 469
3571
1637
ometric value and much lesser in case of CHA [29]. The
3435 962
1482 initial increase in pH may be due to the surface dissolution
1424 631
of HA which releases OH– ions into the solution [30].
599 Figure 8 shows the XRD patterns of the OHA, SHA and
Transmittance (%)

560
1088 CHA samples after the in vitro solubility study. As there is
1046
SHA
no change in the patterns before and after the in vitro study,
this also confirms that all the three HAs are stable in body
fluids. Though the XRD and FTIR show similarity among
the three HAs, the SEM, surface area measurement, and
CHA
in vitro studies clearly indicate the microwave processed
OHA has better properties than SHA and CHA.
The sinterability of OHA has been studied from 800 to
1,250 °C and the results are compared with SHA and CHA
4000 3500 3000 2500 2000 1500 1000 500 samples. The XRD patterns of the OHA pellets sintered
Wave number (cm )
-1 at different temperatures are shown in Fig. 9. The
peak positions in all the patterns from 800 to 1,200 °C
Fig. 4 FT-IR spectra of HA samples

Fig. 5 SEM micrographs of (a)

OHA (b) SHA (c) CHA powder
and fractured surfaces of OHA
(d), SHA (e) and CHA (f)
pellets sintered at 1,200 °C

CHA solubility curve lies below that of SHA and the SHA correspond to the pure HA, where as the XRD pattern of
curve itself lies below that of OHA. As the stoichiometric the pellet sintered at 1,250 °C shows a decrease in the
HA is very stable at pH 7.2, the present results, indicates intensity of the HA and the presence of additional peaks at

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1740 J Mater Sci: Mater Med (2007) 18:1735–1743

350

300

In tens ity (arb. un its)

250
OHA

200

150 SHA

100

50
CHA

0
20 30 40 50 60
Fig. 6 TEM bright field image of OHA powder sample
2 theta (deg)

Fig. 8 XRD pattern of HA samples after dissolution

37.3 and 55.3°, corresponding to the major peaks of cal-
cium oxide (CaO) i.e. (2 0 0) and (2 2 0) (JCPDS 37-1497).
So from the figure it appears that the OHA seems to and sintering studies. The near stoichiometric Ca/P ratio
decompose at around 1,250 °C. The XRD patterns of the observed in OHA may be due to the nature of the calcium
sintered pellets of SHA as a function of sintering temper- precursor. The presence of b-TCP in the CHA even at
ature are shown in Fig. 10. Similar to OHA, SHA is also 900 °C clearly indicates that the Ca/P ratio is much lower
stable upto 1,200 °C but the 1,250 °C pattern shows the than the stoichiometric value [24].
presence b-TCP as the major phase. Figure 11 shows the The variation of density of HA pellets as a function of
XRD patterns of sintered CHA pellets at the above-men- sintering temperature is shown in Fig. 12. The OHA has a
tioned temperatures. The b-TCP appears as a major phase maximum density at 1,200–1,250 °C, which has been
at 900 °C. The allotropic transformation of b-TCP to a- found to be comparable with the reported value for sintered
TCP (9-348 JCPDS) was observed for CHA samples sin- pellets of nanocrystalline HA (98% of the theoretical
tered at 1,250 °C with the appearance of peaks of a-TCP density) [28]. At this temperature, OHA has 98.8% of the
along with b-TCP. The formation of TCP during sintering theoretical density of HA (3.156 g/cm3, JCPDS 9-432),
of SHA and CHA indicates that their Ca/P molar ratio is whereas SHA has 97% of its theoretical density. The
below 1.67. Similarly the presence of CaO during the density of CHA continuously increases with sintering
sintering of OHA indicates that the Ca/P ratio could be temperature and corresponds to the density of b-TCP phase
slightly exceeding 1.67. Although both OHA and SHA (3.07 g/cm3, JCPDS 9-169). The reason for the good
were prepared under similar procedure, they exhibit dif- compactness of OHA and also SHA is due to their parti-
ferent stoichiometric values as evident from dissolution cle morphology i.e., both HAs have spherulite structure.

Fig. 7 Variation in pH of 7.25

phosphate buffer due to the
immersion of HA samples 7.24
CHA
OHA
7.23 SHA

7.22
pH

7.21

7.2

7.19

7.18
0 20 40 60 80 100 120 140 160 180
Time (hours)

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J Mater Sci: Mater Med (2007) 18:1735–1743 1741

C - Calcium oxide β α -- α TCP

C β α
C
C β -- β TCP
β
β β
1250 α β α β β
α α α 1250

1200
Intensity (Arb.Units)

1200

Intensity (Arb.Units)
1100
1100

1000
1000
β β
β
900 β β
900

800
800

20 30 40 50 60
(2 Theta (Degrees)) 20 30 40 50 60
2 Theta (Degrees)
Fig. 9 XRD patterns of the sintered pellets of OHA
Fig. 11 XRD patterns of the sintered pellets of CHA
The density of SHA is less than that of OHA at all sintering
temperatures may be due to porosities associated with it. 1,200 °C. The CHA has a maximum hardness value of
The density of CHA is less than that of SHA as well as 352 Hv at 1,100 °C and gradually decrease to 338 Hv at
OHA and may arise due to decomposition to HA and 1,200 °C. Although SHA seems to have the lowest hard-
b-TCP where density of latter is less. At sintering tem- ness above 1,000 °C compared to that of OHA and CHA, it
perature of 1250 °C the OHA density does not vary even should be noted that CHA has already decomposed and the
with presence of CaO (3.35 g/cm3, JCPDS 37-1497), but hardness corresponds to that of b-TCP. The observed de-
the SHA shows a dip in density due to b-TCP formation. crease in hardness of the OHA at 1,250 °C is due to CaO
The apparent increase in the density of CHA with sintering formation while the decrease above 1,100 °C in case of
temperature even at 1,250 °C is not because of HA but due CHA may be due to phase transformation of b-TCP to
to good sinterability of b-TCP and also due to the allotropic a-TCP [24]. The lower hardness value of SHA may be due
transformation of b-TCP to a-TCP (2.87 g/cm3, JCPDS to porosities. The high hardness of the OHA indicates its
9-348) [24]. good sinterability.
The variation in hardness values of the pellets of the The SEM photographs of the freshly fractured surface of
three HAs with sintering temperature is shown in Fig. 13. the OHA, SHA and CHA pellets sintered at 1,200 °C are
The OHA has a maximum hardness value of 445 Hv at shown in Fig. 5d–f. The fractured surface of OHA shows

β β -- β -TCP
β β β 100
β β
1250 OHA

98
1200 SHA
Intensity (Arb.Units)

Density (% Theoretical)

96
CHA
1100
94

1000
92

900 90

800 88

20 30 40 50 60 800 900 1000 1100 1200 1300

2 Theta (Degrees) Temperature (°C)

Fig. 10 XRD patterns of the sintered pellets of SHA Fig. 12 Density variation of HA pellets with sintering temperatures

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1742 J Mater Sci: Mater Med (2007) 18:1735–1743

520

480
OHA
440

400
% Weight loss

360
CHA

320
SHA
280

240

200

160
800 900 1000 1100 1200 1300
Temperature (°C)

Fig. 13 Hardness variation of HA pellets with sintering temperatures

depressions due to intergranular fracture. The OHA

agglomerates of spherulites as observed in Fig. 5a
increases the compactness of the material, which in turn
favours the high density of the pellet. The fracture surface
Fig. 14 Photographs of OHA (a) and SHA (b) pellets with osteoblast
of SHA exhibits presence of more pores, which are cells
responsible for the lowering its density and hardness val-
ues. The characteristic fracture surface of the CHA clearly
indicates that it is an intragranular fracture. Although the calcium carbonate, arising from the use of eggshell in
XRD analysis of the sintered pellets show that both the synthesis, may play a role in improving the properties of
microwave processed HAs are stable at high temperatures, OHA.
the hardness and density values clearly indicates that OHA
has superior sintering properties than the SHA and CHA.
The results of the qualitative evaluation of the in vitro Conclusions
cytotoxicity test of the apatite samples are listed in Table 2.
The osteoblast cell culture study was carried out on HA Nanocrystalline hydroxyapatite (OHA) has been success-
pellets sintered at 900 °C. Since OHA and SHA were fully synthesized from eggshell waste using microwave
stable at 900 °C, cell culture was evaluated on these irradiation. Compared to the SHA and CHA, the OHA
samples. The MG-63 osteoblast cells around the OHA and seems to have better morphology, stoichiometry, sinter-
SHA samples showed normal response as cells around ability, stability at high temperatures and osteoblast cell
negative control material (Table 2). The presence of more adhesion. The eggshell seems to be a promising source
osteoblast cells was observed on the surface of the OHA of calcium for preparing nanocrystalline hydroxyapatite
compared to SHA after the incubation period of a day as with excellent properties so essential for hard tissue
shown in the micrographs (Fig. 14). Thus OHA has been replacement.
found to be noncytotoxic and favouring adhesion of
osteoblast cells. Overall, the OHA seems to have better
properties compared to SHA. The biological origin of References

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Negative control 0 Noncytotoxic
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OHA 0 Noncytotoxic 5. F.-H. LIN, C.-J. LIAO, K.-S. CHEN and J.-S. SUN, Biomaterials
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