Indian J.Sci.Res.

4(3): 092-099, 2014

ISSN : 0976-2876 (Print)

ISSN : 2250-0138 (Online)

CORRELATION BETWEEN CRYSTALLOGRAPHIC PARAMETERS AND

BIODEGRADATION RATE OF NATURAL HYDROXYAPATITE IN
PHYSIOLOGICAL SOLUTIONS
EBRAHIM KARAMIANa, MAHMOOD REZA KALANTAR MOTAMEDIb, HESAM
MIRMOHAMMADIc, d, PARISA SOLTANIb, AMIRSALAR KHANDANe1
a

Assistant Professor, Department of Materials Engineering, Najafabad Branch, Islamic Azad University, Isfahan, Iran
b
Dental Students Research Center, School of Dentistry, Isfahan University of Medical Sciences, Isfahan
c
Assistant Professor, Dental Materials Research Center, Department of Restorative Dentistry, Isfahan University of Medical
Sciences, Isfahan, Iran
d
Researcher, Department of Cariology Endodontology Pedodontology, Academic Centre for Dentistry Amsterdam (ACTA),
Universiteit van Amsterdam and Vrije Universiteit, Amsterdam, the Netherlands
e

Young Researchers and Elite Club, Khomeinishahr Branch, Islamic Azad University, Isfahan, Iran

ABSTRACT
The aim of present work was to study the biodegradation behavior, which is affected by crystalline size and
crystallinity, of three types of HA such as pure HA (PHA) of bovine bone; biological HA (BHA) and natural HA
(NHA) of scapular bone. The physicochemical properties of these three bioceramics were compared in two different
solutions: a simulated body fluid (SBF) and physiological saline. The results estimated using the scanning electron
microscopy (SEM) observation were used to evaluate surface morphology. In addition, X-ray diffraction (XRD) was
used to analyze phase composition, and transmission electron microscopy (TEM) technique was employed to evaluate
the shape and size of the prepared NHA powder. The functional groups of the prepared NHA were identified using
fourier transform infrared spectroscopy (FTIR). The findings indicate that a decrease in lattice parameters induces
an increase in crystallinity and crystallite size thus decreasing HA dissolution in the biological solutions. Using XRD
and TEM, the HA crystallite size was shown to be 27 nm. SEM and TEM observations showed that nanoparticles are
present in NHA and PHA structures. HA biodegradation reduced in the PHA sample. The NHA sample was observed
to achieve better osseointegration for implant materials owing to increased biodegradation.
KEYWORDS: Biodegradation, Nanoparticles, Hydroxyapatite, Saline Solution, Simulated Body Fluid

Tissue diseases and defects, particularly

bone diseases, are serious health conditions that
directly affect the quality of life of the sufferers.
Ceramics used for this purpose are referred to as
bioceramics.
A combination of hydroxyapatite (HA)
implants serving as osteoconductive scaffold is an
interesting approach for the improvement of bone
defect healing. HA, with the chemical formula
Ca10(PO4)6(OH)2, is the main component of bone
and teeth. This bioceramic has been widely used in
dental materials, constituent implants, and bone
substituent materials because of its excellent
biocompatibility, bioactivity, osteoconductivity,
nontoxicity,
and
anti-inflammatory nature.
Furthermore, HA can directly bond to the bone (Lu
and Leng, 2005). It is manufactured in many forms
and can be prepared as a dense ceramic, powder,
ceramic coating, or porous ceramic as required for
particular applications (Ramli et al., 2011).
Multiple techniques have been utilized for the
preparation of HA powder (Aoki, 1991).
Tissues can be replaced by transplantation
or implantation. The significant advantages of
using bioceramics as implants over transplants are
availability, reproducibility, and reliability. In
addition, they may not cause risk of viral or
bacterial infections to patients. HA has good

Corresponding author

osteoconductivity and is used as bulk ceramics,

coatings, and granules in clinical applications.
Therefore, HA has been widely used in
dental implants, alveolar ridge augmentation,
orthopedics, maxillofacial surgery, etc. New
knowledge of apatite lattice constants (LCs) may
shed light on not only their crystal structure
behavior but also their physicochemical properties
thus facilitating the design and fabrication of
specific biomaterials. Moreover, the properties of
HA ceramics can be improved by controlling
important parameters of powder precursors such as
particle
size,
particle
distribution,
and
agglomeration (Vasconcelos et al., 2001). Further,
emphasis was placed on the relationship between
the structure (or LCs) and the atomic properties of
these apatites.
Advances in high-performance computing
techniques allow the calculation of LCs on the
basis of first principles of quantum mechanics
(Langford and Louer, 1996). HA has a hexagonal
symmetry with unit cell lattice parameters a = 0.95
nm and c = 0.68 nm. Considering its lattice
parameters and symmetry, its unit cell is
considered to be arranged along the c-axis. Unlike
other calcium phosphates, HA does not break down
under physiological conditions. Lattice parameter
measurements are used in many situations to

KARAMIAN ET AL.: CORRELATION BETWEEN CRYSTALLOGRAPHIC PARAMETERS AND..

characterize biomaterials. For instance, knowledge

of lattice parameters can provide information about
the thermal properties of a material, help indirectly
determine the compositions in a solid solution,
measure the strain state, and analyze a defect
structure. Therefore, the lattice structure must be
determined
precisely.
Fortunately,
X-ray
diffraction (XRD) can provide such information to
an accuracy of several significant figures if care is
taken during the experiment and subsequent
analysis.
Osteoconductivity,
solubility,
sinterability, and mechanical reliability of HA can
be promoted by controlling its particle size and
structural morphology (Vasconcelos et al., 2001).
Biodegradable HA-coated implants are
widely used to support tissue regeneration and
healing by degrading the materials and
concurrently replacing the implant coatings with
controlled corrosion rate through the surrounding
tissues (Xu et al., 2009).
The present study was designed to extract
BHA and NHA from human and bovine bones, and
PHA was fabricated by using solgel technique,
which is an advantageous and novel method of
synthesizing fine powders owing to fine
homogeneity, high reactivity of starting materials,
and lower processing temperature (Jallot et al.,
2005). Thereafter, the physicochemical properties
of bioceramics such as pure HA (PHA), biological
HA (BHA), and natural HA (NHA) have been
compared and investigated by using XRD in two
different solutions: simulated body fluid (SBF) and
physiological saline. The aim of the present work
was to investigate the biodegradation behavior,
which is affected by crystallite size and
crystallinity, of the three types of HA (PHA, BHA,
and NHA).

NHA. Similarly, a scapular bone (Al-Zahra

hospital bone bank, Isfahan, Iran) was heated at
900C for 2 h to create BHA. The PHA was created
by using the sol-gel method. Elemental analyses of
the NHA and the BHA were performed with X-ray
fluorescence (XRF) (Bruker S4 PIONEER,
Germany).
An SBF was prepared according to the
procedure described by Kokubo in (Kokubo and
Takadama, 2006). The ion concentrations in the
SBF were similar to those in human blood plasma
(Table 1). The powders were weighed in amounts
of 0.3 g and pressed in a 6-mm diameter steel disc
mold for 2 min under a 9000 N force. Thereafter,
the mixed powders were cold pressed under 600
MPa, and the prepared compact samples were
sintered at 850C for 2 h. The sintered specimens
were cooled slowly in a furnace having a heating
rate of 10C/min. The disc samples were soaked in
a cell SBF solution (pH 7.4) at 37C and a
physiological saline (pH 5.2) for 1, 6, and 14 days
at a solid/liquid ratio of 1 mg/ml without refreshing
the soaking medium. After being soaked for the
predetermined time periods, the disc samples were
rinsed with deionized water to remove the SBF
solutions and dried in an oven for 1 h at 110C.
Elemental analyses of the SBF and the 0.9 wt. %
physiological saline are shown in Table 1.
Table 1: Comparison of nominal ion
concentrations of SBF, physiological saline, and
human blood plasma
Ion

SBF [mmol]

Physiological
Saline
[mmol]

Human
Plasma
[mmol]

Na+

142.0

153

142.0

5.0

5.0

MATERIAL AND METHODS

2+

Mg

1.5

1.5

Preparation of Powders

Ca2+

2.5

2.5

Bovine bones were boiled for 8 h to

remove flesh and fat. The bones were heated at
110C for 2 h to remove moisture. To prevent
blackening with soot during heating, the bones
were cut into approximately 10-mm thick pieces
and heated at 500C (bone ash) for 2 h in air to
allow evaporation of organic substances. The
resulting black bone ash was heated for 3 h at
850C. The powder mixture was then loaded into a
hardened steel bowl along with a ceramic ball of 15
mm diameter. The ball-to-powder weight ratio was
10:1, and the rotational speed was 400 rpm. The
sample was heated for 8 and 10 h. The mass ratio
of balls to reactants was 10, whereby the overall
ball mass was equal to 127 g. The time of
mechanical activation by the planetary ball mill
was selected to be 10 h. This synthesis is called
thermal decomposition of bone resource to create

Indian J.Sci.Res. 4(3): 092-099, 2014

147.8

153

103.0

4.2

27.0

1.0

1.0

0.5

0.5

7. 4

5.2

7. 27. 4

Cl

HCO3
HPO4
SO4
pH

KARAMIAN ET AL.: CORRELATION BETWEEN CRYSTALLOGRAPHIC PARAMETERS AND..

Table 2: HA biodegradation properties of all the

samples in biological solutions

microscopy (TEM) technique (Philips 100 KV

EM208S).
The crystallite size of the milled powder
was determined by using XRD pattern and the
modified Scherrer equation approach (Monshi et
al., 2012). In addition, Fourier transform infrared
spectroscopy (FTIR) was employed to identify the
functional groups of the prepared NHA.

WL. % of
Samples in
SBF after
2 Weeks
(mg)

WL. % of
Samples
in Saline
Solution
after 2
Weeks
(mg)

Ca2+ Release
in SBF after
2 Weeks
(ppm)

Ca2+
Release in
Saline
Solution
after 2
Weeks
(ppm)

PHA

0.39

1.06

48

61

In Vitro Evaluation

BHA

0.62

1.67

55

68

NHA

3.31

4.02

62

115

In vitro bioactivity evaluation of the

synthesized HA powder was performed in an SBF
medium of 7.4 pH at a ratio of 1 mg/ml in a water
bath at 37C. SEM was used to identify apatite
formation on the surface of the samples and
evaluate the surface morphology of the samples
after immersion in the SBF medium. Studies have
been conducted on nanobioceramic range by
monitoring the behavior of a material when
submersed in a saline solution (Sridhar et al., 2003)
or an SBF (Kokubo and Takadama, 2006).

Sample

Table 3: Crystallographic parameters of HA

phase in all the samples
Sample

Lattice
Par. (A)

Crystallinity
Xc (%)

Crystallite
Size (nm)

PHA

9.417

97.1

53,4

BHA

9.425

90.2

44.3

NHA

9.443

84.6

27

Table 4: Average HA crystallite size in

comparison with c/a ratio
Sample

ln(k/L)

Ave. L (nm)

c/a Ratio

PHA

5.959

53.4

0.742

BHA

5.779

44.3

0.736

NHA

5.225

27

0.733

In this in vitro evaluation, the nanopowder

was investigated by soaking the powder in the SBF
and the physiological solution. After soaking for
the intended time periods, the disc samples were
rinsed with deionized water and dried in an oven at
110C for 1 h. The weight loss of the samples was
calculated by using equation 1.
Weight loss (WL. %) = (W W0) / W0 100, (1)
Where W and W0 are the primary and
secondary weights, respectively.
The lattice parameters are calculated by
using equation 2.
1/d2 = (h2 + hk + k2)/a2 + l2/c2

Characterization Procedure
Phase structure analysis was carried out by
using XRD analysis with a Philips XPert MPD
diffractometer using Cu Ka radiation (1 = 0.15418
nm) over a 2 range of 2080. The obtained
experimental patterns were compared with the
standard patterns compiled by the Joint Committee
on Powder Diffraction and Standards (JCDPS),
which involved card no. 09-432 for HA. Scanning
electron microscopy (SEM) evaluations were
performed with a Philips XL30 (Eindhoven, The
Netherlands)
to
investigate
the
surface
morphology. The Ca/P ratio was determined by
using
energy-dispersive
X-ray
(EDX)
spectroscopic microanalysis. The samples were
coated with Au by spraying in high vacuum at an
accelerating voltage of 25 kV. The Ca and P
contents were measured at four positions and then
averaged. The shape and size of the prepared NHA
was evaluated by using the transmission electron

Indian J.Sci.Res. 4(3): 092-099, 2014

(2)

Where d is the distance between the planes with

Miller indices h, k, and l.
In addition, crystallinity (Xc) is calculated
using XRD data and equation 3 as follows:
Xc = 1 (V112/300/I300)

(3)

Where I300 is the intensity of the (300)

diffraction peak, and V 112/300 is the intensity of the
hollow between the (112) and the (300) diffraction
peaks of the produced powder.

RESULTS
General Findings
The lattice parameters and the crystallinity
and weight loss values of the HA-based samples
and Ca2+ released in solutions are shown in Tables
2 and 3. Results indicate that during 1, 6, and 14
days of soaking, Ca2+ ions are released into the
solution (Table 2). Table 4 shows the values of
crystallite size and the c/a ratios of HA phase in the
samples. The variations in pH values of the SBF

KARAMIAN ET AL.: CORRELATION BETWEEN CRYSTALLOGRAPHIC PARAMETERS AND..

medium were measured at predetermined time

intervals using a pH meter (Fig. 1).

7.5

7.45
7.4

pH (ppm)

7.35
7.3

PHA

7.25

BHA

7.2
7.15

NHA

7.1
7.05
7
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (Day)

Figure 3: Plot of ln vs. ln (1/Cos ) of NHA

sample.

Figure 1: pH vs. soaking time curves of (a) SBF

and (b) saline solutions.

Figure 4: FTIR spectrum of the NHA sample

obtained by heating at 850C for 3 h.

Figure 2: XRD patterns of BHA, NHA, and

PHA samples.

Indian J.Sci.Res. 4(3): 092-099, 2014

KARAMIAN ET AL.: CORRELATION BETWEEN CRYSTALLOGRAPHIC PARAMETERS AND..

Figure 5: SEM micrographs of the prepared (a) BHA, (b) PHA, and (c) NHA nanopowders.

Figure 6: TEM micrographs (100 and 50 nm) of the prepared NHA powder obtained after milling bovine
bone for 10 h.

As shown in Table 5, the Ca/P weight ratios are 2.81 and 2.74 for NHA and BHA, respectively.

Indian J.Sci.Res. 4(3): 092-099, 2014

KARAMIAN ET AL.: CORRELATION BETWEEN CRYSTALLOGRAPHIC PARAMETERS AND..

Table 5: Semi-quantitative XRF analysis results of NHA and BHA

XRF Analysis Results

NHA

BHA

Compound

Concentration
(%w/w)

Compound

Concentration
(%w/w)

Ca

76.91

CaO

60.94

23.09

P2O5

36.37

Na

1.25

MgO

0.830

1.20

Na2O

0.696

Mg

0.708

SO3

0.284

Sr

0.443

SiO2

0.271

Cl

0.200

SrO

0.197

Si

0.150

Al2O3

0.060

0.073

Cl

0.057

Al

0.061

Fe2O3

0.038

Cu

0.056

CuO

0.031

Zn

0.053

ZnO

0.021

Fe

0.052

K2O

0.020

0.038

--

--

Zr

0.008

--

--

LOI*

0.376

LOI

0.131

Total

100.05

Total

99.95

* Loss on ignition (1000 C, 2 h)

Phase and Composition Analyses of XRD

Results
Figure 2 shows the XRD pattern of the
NHA. This indicates that the only existing phase is
for HA (i.e., all the peaks belong to HA). HA was
synthesized by 10 h of mechanical alloying, and the
biological apatite of the bone indicates that the HA
was synthesized similar to the biological apatite of
the bone. The c/a ratio (lattice parameters) was
calculated from the XRD data (Table 4). The data
indicate that a decrease in lattice parameters
induces an increase in crystallinity and crystallite
size thus decreasing HA dissolution in the
biological solutions. The crystallite size of the
milled powder was determined by using the XRD
pattern and the modified Scherrer equation
(Monshi et al., 2012), and is equivalent to ln =
ln(1/cos ) + ln(k/L). The modified Scherrer
equation provides the advantage of decreasing the
sum of absolute values of errors,(ln)2, and
produces a single line through the points to give a

Indian J.Sci.Res. 4(3): 092-099, 2014

* Loss on ignition (1000oC, 2 h)

single value of intercept ln(k/L). For this sample,
the linear regression plot is obtained as y = 0.9267x
5.225. For this line, the intercept is 5.225, and
e5.225 = k/L and L= 27 nm. Therefore, the average
HA crystallinity size was 27 nm. The modified
Scherrer equation indicated eight plots of ln vs.
ln(1/cos ) for the XRD pattern of the NHA
sample. Figure 3 shows eight plots of ln vs.
ln(1/cos ) for the XRD pattern of the NHA sample
along with the equations of linear least-squares
method obtained from linear regression of data in
Excel plots. The slopes are positive because at
higher angles of 2, with lower values of cos and
higher values of ln(1/cos ), the observed and
measured values are less than those expected
with the modified Scherrer equation. Therefore, for
the linear plots, a slope of 45 can rarely be
achieved. The errors involved in the modified
Scherrer equation when employing different peaks
primarily cause the scattering of the points. Other
sources of error include ln and ln(1/cos )
measurements. The significance of this work is to

KARAMIAN ET AL.: CORRELATION BETWEEN CRYSTALLOGRAPHIC PARAMETERS AND..

minimize the errors by applying a method that uses

the least-squares technique for obtaining accurate
results. Moreover, although variations exist in ln
values, the intercept varies systematically as
5.225, 5.779, and 5.959 for NHA, BHA, and
PHA, respectively. In the other words, HA
crystallite size averages are 27, 44.3, and 53.4 nm,
respectively. Figure 2 shows the XRD patterns of
the PHA, BHA, and NHA samples. As can be seen,
in all the samples, the only existing phase is for
HA.
FTIR Evaluation
Fourier transform infrared spectroscopy
(FTIR) is a measurement technique in which the
spectra are collected on the basis of measurements
of the coherence of a radiative source using time
domain. Figure 4 shows the FTIR spectrum of the
NHA sample obtained by heating at 850C for 3 h.
It illustrates the presence of covalence bonds such
as PO43 and OH. This spectrum and the XRD
result (Fig. 2) confirm the presence of the
Ca10(PO4)3(OH)2 compound in the NHA sample.
SEM Analysis
SEM observations showed that BHA has
nonspheroid crystals; however, the crystal sizes of
PHA and NHA are in the order of microns, and
some particles are spherical whereas others have
sharp shapes (Fig. 5). The agglomerated particles
are composed of very fine particles.
TEM Analysis
Figure 6 shows the TEM photomicrograph
of
the
NHA nanopowder.
The
TEM
photomicrograph shows relatively uniform
distribution of the nanoparticles in the NHA. As
can be seen in the TEM image (Fig. 6), after 10 h
of milling, the prepared NHA powder has an
average crystallite size similar to that obtained with
the modified Scherrer equation. In addition, the
crystallite size of the NHA sample was similar to
that of the BHA sample. The TEM image reveals
that the powders are formed by particles ranging
from 50 to 100 nm.

DISCUSSION
In the present study, the gradual increase
in the lattice parameters suggests a decrease in
crystallinity values, leading to a reduction in
weight loss of the specimens and Ca2+ release in
the solutions (Table 2). SEM was used to identify
apatite formation on the surface of the samples and
evaluate the surface morphology of the samples
after immersion in the SBF medium. The SEM
observations showed that the BHA has nonspheroid
crystals. However, the crystal sizes of PHA and
NHA are in the order of microns, and some
particles are spherical whereas others are sharp
shaped (Fig 5). In fact, HA dissolution was reduced

Indian J.Sci.Res. 4(3): 092-099, 2014

by increasing the amount of crystallinity and

decreasing lattice parameters. As shown in Table 2,
greater weight loss of samples and calcium release
in the solutions (HA dissolution) occurred in the
physiological saline than in the SBF within all the
samples. The presence of higher levels of chloride
(Cl) and Na+ ions in the physiological saline (153
mmol) than in the SBF (147 mmol and 142 mmol)
may increase HA dissolution (Table 1). Moreover,
the presence of Cl ions resulted in degradation of
HA crystals in the samples. The NHA powder
exhibited greater surface area and is expected to
have better bioactivity than coarser crystals (Stupp
and Ciegler, 1992). The rate of Ca2+ ions releasing
from the PHA into SBF indicated that the pattern of
calcium release in NHA was similar to that in BHA
(Table 2). Moreover, HA biodegradation was
reduced in the PHA samples. In fact, weight loss
and Ca2+ ions release were the least in these
samples. The results showed that with an increase
in soaking time of the powders in the SBF solution
for up to two weeks, calcium release was increased.
The results estimated from the XRD data indicate
that a decrease in lattice parameters induces an
increase in crystallinity, which decreases HA
dissolution in biological solutions. The average
crystallite size of the produced NHA after 10 h of
milling was approximately 4050 nm with the
modified Scherrer equation approach (Monshi et
al., 2012).
From the TEM image (Fig. 6), the
prepared NHA powder after 10 h of milling was
shown to have an average crystallite size similar to
the determined size obtained with the modified
Scherrer equation. The release of calcium ions
from the NHA into the SBF was quantitatively
estimated to support its in vitro bioresorbability
(Table 2). The results show that more calcium ions
were released from the NHA when compared with
PHA. The amount of calcium released from the
prepared NHA was in agreement with the calcium
release pattern of the biological apatite reported by
other studies (Murugan and Ramakrishna, 2005).
As shown in Figure 1, the pH of the PHA did not
show significant variations because it was not
resorbed in the physiological solutions thus
revealing its physiological stability during the
period of study. However, the NHA powder
showed a dramatic downward variation in pH
suggesting that it dissolves much faster than BHA.
The pH value depends on the solubility or
bioresorbability of the HA, namely, as pH
decreases,
solubility increases.
The
pH
measurement results show a decrease in the pH of
the SBF solution with an increase in soaking time.
Accordingly, as can be seen clearly from Table 2,
the rate of bioresorbability of the prepared NHA is
higher than that of PHA and is similar to that of the
BHA of the bone. Depending on clinical needs, the
solubility of HA can be altered by controlling its

KARAMIAN ET AL.: CORRELATION BETWEEN CRYSTALLOGRAPHIC PARAMETERS AND..

crystallite size. The synthesized NHA had

acceptable functional properties because of its
crystallite size and in vitro properties being similar
to BHA, which would have a significant impact on
implant-cell interaction in a body environment. The
bioresorbability of the synthesized NHA powder
was also similar to that of the biological apatite of
(Murugan and Ramakrishna, 2005).
Further, NHA possesses exceptional
biocompatibility and bioactivity properties with
respect to bone cells and tissues, probably because
of its similarity to the hard tissues of the body.
Further, the NHA is able to improve contact
reaction and stability at the artificial/natural bone
(bovine bone) interface for medical applications
(Murugan and Ramakrishna, 2005).
Control of the crystallite size is a suitable
parameter for tailoring the behavior of HA powders
in dental and orthopedic applications. Therefore,
the prepared NHA was concluded to be more
useful for treatment of oral bone defects when
compared with PHA and can be more effective as a
bone replacement material to promote bone
formation.
In Conclusion
1) HA biodegradation was minimal in the PHA
samples. In fact, the weight loss was the least in
these samples.
2) Greater weight loss of samples and calcium
release in the solutions (HA dissolution)
occurred in the physiological saline than in the
SBF within all the samples. The presence of high
levels of Cl in the physiological saline (153
ppm) and the SBF (147 ppm) leads to the
increase in HA dissolution. HA dissolution was
reduced by increasing the amount of crystallinity
and decreasing lattice parameters.
3) Biodegradation increased in the NHA sample,
which can be considered for achieving better
osseointegration for implant materials.

ACKNOWLEDGMENTS
The authors would like to extend their
gratitude for the support provided by the Najafabad
and Khomeinishahr branches of Islamic Azad
University, Isfahan, Iran.

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