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International Journal of Engineering & Technology IJET-IJENS Vol:14 No:01
24
Preparation and Characterization of GelatinHydroxyapatite Composite for Bone Tissue
Engineering
Md. Jakir Hossana, M. A Gafurb, M. R Kadirb and Mohammad Mainul Karima,*
a
Department of Applied Chemistry and Chemical engineering, University of Dhaka, Dhaka-1000, Bangladesh
b
PP & PDC, Bangladesh Council of Scientific and Industrial Research (BCSIR ), Dhaka, Bangladesh
*To whom correspondence should be addressed: Mohammad Mainul Karim
E-mail: [email protected]
E-mail: [email protected] or [email protected] Web Site: http://www.ijens.org
Abstract--
In biomedical research, fabrication of porous
scaffolds from advanced biomaterial for healing bone defects
represents a new approach for tissue engineering.
Hydroxyapatite ceramics have been recognized as substitute
material for bone and teeth in orthopeadic and dentistry field
respectively due to their chemical and biological similarity to
human hard tissue. In this study, to mimic the mineral and
organic component of natural bone, hydroxyapatite (HAp) and
gelatin (GEL) scaffolds were prepared. The raw material was
first compounded and resulting composite were molded into the
petridishes. Using Solvent casting process, it is possible to
produce scaffolds with mechanical and structural properties
close to natural trabecular bone. The chemical and thermal
properties of composites were investigated by Fourier Transform
Infrared Spectroscopy (FTIR), Thermogravimetric Analyzer
(TGA),
Differential
Thermal
Analyzer
(DTA)
and
Thermomechanical
Analyzer
(TMA).
Crystallographic
characterization by X-Ray diffraction and morphological
characterization by SEM revealed the formation of a micro
porous hydroxyapatite gelatin composite. It was observed that
the pores in the scaffolds are interconnected and their sizes range
from 80 to 400μm.
Since one osteoblast occupies an area of approximately 700 μm,
hence the pore size of 500 μm (diameter of a spherical pore) is
compatible with osteoconduction, however the optimum pore size
for osteoconduction is 150 μm. These results demonstrate that the
prepared composite scaffold is a potential candidate for bone
tissues engineering.
1. INTRODUCTION
Tissue
engineering
is
an
interdisciplinary
and
multidisciplinary field that aims at the development of
biological substitutes that restore, maintain, or improve tissue
function [1]. In a typical tissue engineering approach, to
control tissue formation in three dimensions (3D), a
considerable porous scaffold is critical. In addition to defining
the 3D geometry for the tissue to be engineered, the scaffold
provides the microenvironment (synthetic temporary
extracellular matrix) for regenerative cells, supporting cell
attachment, proliferation, differentiation, and neo tissue
genesis [2]. Therefore, the chemical composition, physical
structure, and biologically functional moieties are all
important attributes to biomaterials for tissue engineering. The
most important advances in the field of biomaterials over the
past few years have been in bioactive biomaterials. Tissue
engineering presents an alternative approach for the healing of
diseased, damaged and traumatized bone tissue. In a typical
tissue engineering application, osteogenic cells would be
harvested from the patient and seeded on a synthetic porous
(structure having a large surface area for a more efficient cell
interaction) scaffold that acts as a guide for tissue growth
creating a living biocomposite. The biocomposite system
would then be implanted back into the patient. Eventually, the
scaffold will be absorbed by the body as non-toxic
degradation products at the same rate that the cells produce
their own extracellular matrix. One of the major challenges of
tissue engineering is to develop a suitable bone scaffold. The
primary concerns are biocompatibility, biodegradability, pore
size, pore connectivity and an adequate mechanical strength.
Bone tissue engineering has the potential to reach millions
annually to repair the bone defects caused by diseases, trauma
or congenital defects. In 2003 the potential market for tissue
engineered products for musculoskeletal applications totaled
23.3 billion in the US and is expected to rise to 39 billion by
the year of 2013[3].
In 2004 alone there were 1.3 million bone grafts procedures
[4]. In the united states alone, at least eight million surgical
operations were carried out annually, requiring a total national
healthcare cost exceeding 400 billion per year [5,6].
Autografts (from the patient) were considered the gold model
for bone defects and allografts ( from the donor were also
commonly used). Commonly used materials for this purpose
are biodegradable polymers such as poly(glycolic acid)
(PGA), poly(lactic acid) (PLA) and their copolymers
(PLGA).[7–10] For example, PLA degrades within the human
body to form lactic acid, a naturally occurring substance
which is easily removed from the body material. Scaffolds
based on these two polymers have been used in numerous
tissue engineering applications [5]. However, most polymers
have relatively poor mechanical strength which is required for
many applications [11–14]. A common way to improve the
mechanical properties of polymers is to make use of filler
particles. Thus, in order to obtain a better combination of
biocompatibility, biodegradation and mechanical strength,
composites of polymer and bioactive ceramics have been
considered for bone tissue engineering [15–18].
Hydroxyapatite (HAp), owing to its excellent bioactivity,
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International Journal of Engineering & Technology IJET-IJENS Vol:14 No:01
osteoconductivity and chemical similarity to the mineral
component of natural bone, has been a preferred bioceramic in
the fabrication of composite scaffolds [7,19,20]. It should be
added that polymer ceramic system resembles the natural bone
structure which itself is a composite composed of nanosized
hydroxyapatite embedded in collagen matrix. The biological
response to the scaffold material is influenced by a number of
factors including the size and morphology of the pores within
the fabricated scaffold [21,22]. The optimum pore size is
different depending on the cell type. However, generally an
interconnected pore structure with a pore size in the range of
100–500 mm is considered to be optimal for osteoconduction
and nutrient transfer for optimal tissue growth [23,24].
Various processing methods have been adopted in the
preparation of porous scaffolds including gas foaming [7],
freeze drying [9,25], casting methods and porogen particulate
leaching [26–28].
Among these techniques, casting methods seems to be a rather
simple and economical approach in the preparation of porous
scaffold materials. In this casting method, the scaffold’s
porosity can be controlled by the amount of a water soluble
leaching agents such as sodium chloride, while the pore size
can be manipulated by the size of the salt crystals [29,30].
There are some recent reports on the use of this procedure in
the preparation of porous (PLGA) and HAp–PLA scaffolds.
This method has also been applied in the fabrication of porous
gelatin-HAp scaffold [29]. Due to its biodegradability,
biocompatibility and cost efficiency, gelatin, a natural
polymer can be used as a scaffold for tissue engineering.
However, as it was mentioned previously, scaffolds based
upon polymeric material alone such as gelatin are not ideal in
terms of their mechanical strength [29]. To address this issue
and to keep a proper balance between the biological and the
mechanical strength, the addition of bioactive HAp particles
within a gelatin matrix has been reported in the literature [25].
The purpose of this study was therefore to extend into the
preparation of hydroxyapatite–gelatin composites and to
investigate the structure.
2.
25
white coloration during preparation is an indication for the
formation of hydroxyapatite[32]. After reaching this point, the
suspension was stirred for 2 h, before it was left undisturbed
for 24 h at room temperature. The HAp precipitates were then
separated from suspension by filtration. At some times the wet
cakes were washed with hot distilled water and dried in a
dryer at 1000C. Then it was powdered in a mortar with pestle.
2.2 Preparation of Gelatin – HAp Composite
The slurry composite was prepared using solvent casting
method. As dry GEL is essentially intractable material, it can
readily become castable or shapeable when transformed into a
sol-GEL state by dissolution in water up to about 5-30 wt% .
In order to have a homogenous and strong composite, the HAp
particles finer than 150 μm were obtained using
Ultrasonicator. Definite GEL content 12.33 wt% was
dissolved in dionized water at temperature of 45°C. Then the 5
wt%, 10 wt%,15 wt% and 20wt% HAp contents were added to
prepare four different composites. The reinforced slurry
composite was then heat treated on magnet stirrer under
constant mixing at 40°C for 1 h. The slurry was
deagglomerated by magnet stirring. The temperature was
monitored continuously. It should be noted that to make a well
distributed homogenous composite, the heat must not be
applied directly to the composite.
By using an interface water bath beaker, the heat treatment
process can be homogenously applied to the reaction vessel.
Some air bubbled were generated which was removed by glass
rod. The slurry immediately was transferred into definite size
of petridishes. The molds were frozen at - 40°C and then dried
in a commercial freeze-dryer for 3 h for solvent removal.
After that, the white composites were removed and placed in
room temperature for 24 h. For statistical analysis in all
assays, five samples of each type were investigated and the
average was reported.
3.
RESULTS AND D ISCUSSION
3.1 TGA/DTA/ DTG analysis for Gelatin –HAp Composite
MATERIALS AND METHOD
2.1 Synthesis of HAp Powder
Preparation of gelatin–hydroxyapatite composite scaffold
started by preparing hydroxyapatite crystallites using a
chemical precipitation method [31]. At first 1M Ca(OH) 2
(96% pure) was taken with 100 ml distilled water in a beaker.
On the other hand 0.6 M H3PO4 was taken with 100ml
distilled water in another beaker. Appropriate amounts (Ca/P
ratio of 1.67) of orthophosphoric acid solution was added
gradually (2 drops per second) from burette in the beaker with
magnetic stirrer at room temperature. The appearance of milky
TGA tell the physical properties of the polymer used in the
scaffold preparation. TGA shows the change in mass with the
increase of temperature. TGA/DTA and DTG studies was
carried out by TG/DTA 6300, SII Nano Technology, Japan,
system controlled by an EXSTAR 6300 controller. TGA and
DTA studies have been carried out on Gelatin-HAp composite
sample in different weight. Experiments have been performed
using simultaneous TGA-DTA analysis by heating the sample
at 20 Cel/min in the temperature range 0˚C and 600˚C in
nitrogen atmosphere and a typical plots has been shown in
figures 1-5.
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International Journal of Engineering & Technology IJET-IJENS Vol:14 No:01
1.4%
111.2Cel
125.9Cel
97.6%
94.6%
100.0
25.00
250.0
26
294.1Cel
89.8%
126.7Cel
139.8Cel
6.4%94.4%
91.2%
90.0
80.0
20.00
332.8Cel
67.7%
200.0
70.0
44.1% 326.3Cel
71.3%
60.0
50.0
371.2Cel
45.6%
10.00
100.0
TG %
150.0
DTA uV
DTG ug/min
15.00
40.0
320.8Cel
8.69uV
30.0
5.00
20.0
326.5Cel
53.2ug/min
50.0
126.0Cel
19.5ug/min
10.0
0.00
0.0
0.0
-10.0
100.0
200.0
300.0
Temp Cel
400.0
500.0
Fig. 1. TG, DTA and DTG curves for Pure Gelatin.
TG (figure-1) shows the 1.4% initial loss due to moisture. The
first onset temp, 2nd onset temperature, first 50% degradation
temperature, 2nd 50% degradation temperature,1st maximum
slope, 2nd maximum slope are 111.20C, 294.10C, 125.90C,
332.80C, 126.7oC and 326.3oC respectively. The total
700.0
100.0
291.1Cel
93.1%
25.00
127.0Cel
97.1%
800.0
degradation loss is 44.1%. DTA curves shows endothermic
peaks at 320.80C are due to thermal degradation. The DTG
curves show the two peaks at 126.00C and 326.50C. There is
two steps degradation, the initial degradation is due to
moisture and the 2nd degradation is due to composite.
330.1Cel
68.3%
80.0
20.00
48.8%
600.0
329.6Cel
68.7%
60.0
15.00
368.2Cel
44.3%
40.0
547.7Cel
41.0%
400.0
TG %
DTA uV
DTG ug/min
500.0
10.00
323.0Cel
9.85uV
300.0
20.0
5.00
200.0
0.0
328.6Cel
182.0ug/min
100.0
0.00
-20.0
0.0
100.0
200.0
300.0
Temp Cel
400.0
500.0
600.0
Fig. 2. TG, DTA and DTG curves for gelatin +5% HAp Composite.
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The onset temperature (figure-2), 50% degradation
temperature, maximum slope are 291.10C, 330.10C, and
329.6oC respectively. The total degradation loss is 48.8%.
DTA curves shows endothermic peaks at 323.0 0C are due to
27
thermal degradation. The DTG curves shows the one peak at
328.60C . There is one step degradation is due to thermal
degradation of composite.
128.2Cel
95.3%
100.0
800.0
40.00
4.3%
158.0Cel
94.1%
287.1Cel
93.6%
331.1Cel
69.5%
700.0
80.0
30.00
331.3Cel
48.2% 69.4%
600.0
60.0
373.3Cel
45.3%
544.2Cel
41.5%
400.0
40.0
TG %
20.00
DTA uV
DTG ug/min
500.0
10.00
300.0
200.0
20.0
329.4Cel
198.1ug/min
0.00
0.0
100.0
-10.00
-20.0
0.0
100.0
200.0
300.0
Temp Cel
400.0
500.0
600.0
Fig. 3. TG, DTA and DTG curves for gelatin +10% HAp Composite.
TG (figure-3) shows the 4.3% initial loss due to moisture The
onset temperature, 50% degradation temperature, maximum
slope are 287.10C, 331.10C, and 331.3oC respectively. The
total degradation loss is 48.2%. DTA curves shows
140.4Cel
95.0%
40.00
800.0
35.00
4.4%
700.0
endothermic peaks at 338.10C are due to thermal degradation.
The DTG curves shows the one peak at 329.40C . There is one
step degradation is due to thermal degradation of composite.
100.0
289.3Cel
93.7%
167.5Cel
94.2%
90.0
333.5Cel
71.3%
80.0
30.00
600.0
342.2Cel
44.9%
66.9%
25.00
70.0
60.0
50.0
378.7Cel
48.8%
400.0
575.5Cel
47.8%
15.00
TG %
20.00
DTA uV
DTG ug/min
500.0
40.0
300.0
10.00
200.0
5.00
100.0
0.00
30.0
375.1Cel
10.62uV
20.0
10.0
331.3Cel
167.4ug/min
0.0
-5.00
0.0
-10.0
100.0
200.0
300.0
Temp Cel
400.0
500.0
600.0
Fig. 4. TG, DTA and DTG curves for gelatin +15% HAp composite.
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endothermic peaks at 375.10C are due to thermal degradation.
The DTG curves shows the one peak at 331.3 0C. There is one
step degradation is due to thermal degradation of composite.
TG (figure-4) shows the 4.4% initial loss due to moisture The
onset temperature, 50% degradation temperature, maximum
slope are 289.30C, 333.50C, and 342.20C respectively. The
total degradation loss is 44.9%. DTA curves shows
129.1Cel
94.9%
700.0
30.00
100.0
284.1Cel
97.2%
157.2Cel
94.8%
90.0
330.6Cel
75.5%
4.5%
600.0
28
25.00
80.0
43.4%
500.0
331.8Cel
74.9%
70.0
20.00
15.00
372.3Cel
53.8%
526.4Cel
51.1%
50.0
TG %
400.0
DTA uV
DTG ug/min
60.0
40.0
300.0
10.00
322.7Cel
10.34uV
376.0Cel
10.93uV
30.0
200.0
5.00
100.0
20.0
10.0
332.8Cel
185.6ug/min
0.00
0.0
0.0
-10.0
-5.00
100.0
200.0
300.0
Temp Cel
400.0
500.0
600.0
Fig. 5. TG, DTA and DTG curves for gelatin +20% HAp composite.
TG (figure-5) shows the 4.5% initial loss due to moisture The
onset temperature, 50% degradation temperature, maximum
slope are 284.10C, 330.60C, and 331.8oC respectively. The
total degradation loss is 43.4%. DTA curves shows
endothermic two peaks at 322.70C and 376.00C are due to
initial and final thermal degradation respectively. The DTG
curves shows the one peak at 332.80C . There is one step
degradation is due to thermal degradation of composite.
are due to thermal degradation. From the TG/DTA data it is
observed that HA-GEL composite is highly stable. The
stability of composite increases with increasing % of HA.
TGA and DTGA show that all the samples exhibited three
⁰
⁰
distinct weight loss stages at 40 C-250 C (5% weight loss of
⁰
⁰
weakly physioabsorbed water), 250 C-500 C (decomposition
of main chain of gelatin). Nevertheless major weight losses
⁰
⁰
are observed about 50wt% in the range of 250 C-500 C for all
the samples, which are corresponding to the structural
decomposition of gelatin.
First order derivative of TGA curves reveals the temperature
at which the maximum decrease of mass occur. The
⁰
temperature at the maximum loss rate is 326.5 C for the pure
⁰
⁰
Gelatin, 328.5 C for the 5wt% HA-GEL composite, 329.4 C
o
for the 10wt% HA-GEL composite, 331.3 C for the 15wt%
Ha- GEL composite and 332.8oC for the 20wt% HA- GEL
composite. DTGA data clearly show that endothermic peaks
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3.2 X-Ray diffraction analysis:
Fig. 6. X-Ray diffraction spectra of pure HAp, Pure Gelatin, 5wt%, 10wt%, 15wt%, and 20wt% of HAp respectively (from top).
The phase of the gelatin and gelatin-HA composite was
analyzed with XRD patterns, as shown in Figure 6. In pure
gelatin, a broad peak at 2θ ≈ 14°, the characteristic of gelatin,
was observed. When HA was added, typical HA peaks were
observed, and with increasing HA amount, the peak intensities
increased and the gelatin peak decreased correspondingly.
3.3 Fourier Transform Infrared (FTIR) analysis:
Fig. 7. FTIR spectrum of Pure Gelatin
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Fig. 8. FTIR spectrum of HAp- Gelatin composite at containing 5% HAp
Fig. 9. FTIR spectrum of HAp- Gelatin composite at containing 10% HAp.
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The FTIR spectrum of the Gelatin and 5 wt%, 10 wt% HApGelatin composites are shown in Figure-7-9. The band at 1328
cm-1 in GEL is attributed predominantly to the so-called
wagging vibration of proline side chains. The 1328 cm-1 band
in GEL does not simply represent the carboxyl group, but it is
one of a number of bands in the range of 1400- 1260 cm-1
which are attributed to the presence of type-I GEL [1, 2]. The
amide A band arising from N-H stretching was distributed at
3270-3370 cm-1 relative to the degree of cross-linking, C-H
stretching at ~2947 cm-1 for the amide B, C = O stretching at
1637 cm-1 for the amide I, N-H deformation at 1500-1550 cm-1
for the amide II [1,2]. The appearance of an amide I mode
indicated that HA-GEL composites adopt a predominantly αhelical configuration and this is confirmed by the appearance
of amide II at ~1540 cm-1 [3,4]. As HA related bands, there
31
are hydroxyl group (-OH) stretching (4000-3200 cm-1) and
liberational bands, and phosphate contours. There are CO 3 V3
bands at 1540-1400 cm-1 and 1530-1320 cm-1. The phosphate
band is between 900 and 1200 cm-1. The shift of the 1328 cm1
band in GEL has been effectively used to confirm the
chemical bond formation between carboxyl ions in GEL and
HAp phases [1,3]. During the process of HA-GEL composite,
the Ca2+ ions will make a covalent bond with R-COO- ions
of GEL molecules. Moreover the cross-linking induces the
shortening of the distance between HAp-GEL fibrils within
the critical length and more amount of Ca2+ ions on HAp will
have a chance to bind with R-COO- ions of GEL molecules
[2-4].
3.4. Scanning electron microscope analysis:
(a)
(b)
(c)
Fig. 10. SEM image of microstructure of Gelatin-HAP composite with 5% HAp.
(a) at 140 magnification. (b) at 330 magnifications. (c) at 4300 magnification.
(a)
(b)
(c)
Fig. 11. SEM image of microstructure of Gelatin-HAP composite with 10% HAp.
(a) at 65 magnification. (b) at 950 magnifications. (c) at 2300 magnification.
(a)
(b)
(c)
Fig. 12. SEM image of microstructure of Gelatin-HAP composite with 15% HAp.
(a) at 350 magnification. (b) at 1500 magnifications. (c) at 5500 magnification.
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Porosity characterization is based on the presence of open
pores which are related to properties such as permeability and
surface area of the porous structure. It was found from SEM
image analysis as shown in figure 10-12 that the addition of
HAp results in more dense and thicker pore walls with lower
porosity, therefore the addition of HAp content improves the
mechanical properties [5, 6]. Since a higher density of a
scaffold usually leads to higher mechanical strength while a
high porosity provides a favorable biological environment, a
balance between the porosity and density for a scaffold must
be established for the specific application.
[7]
CONCLUSION
Gelatin and Hydroxyapatite (HAp) is one of the components
most frequently used to prepare calcium phosphate
composites, because of its biocompatibility, biodegradation
and innocuousness. In this work we try to prepare and
characterize gelatin & hydroxyapatite using TG/ DTA, SEM
& FTIR analysis and X-Ray diffraction (XRD).
Morphological investigation showed that the HAp particles
exhibit micro-porous morphology, which provides enlarged
interfaces being a prerequisite for physiological and biological
responses and remodeling to integrate with the surrounding
native tissue. XRD analysis revealed the exact crystalline
structure of the three samples is one of the calcium-phosphate
polymorph with Ca/P = 1.65 indicating the formation of
calcium-hydroxyapatite phase. All the four samples exhibited
almost similar diffraction pattern with characteristic peaks of
HAp. The FTIR spectrum for the crosslinked composite
indicates chemical bond formation between gelatin and
hydroxyapatite. From the TG/DTA data it is observed that
gelatin – HAp is highly stable. The degradation temperature of
gelatin nearly 3000C of composite. This study indicates that,
gelatin and HAp can be used as a bone replacement material.
[16]
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