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Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619
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Evaluation of Polyether sulfone/nanohydroxyapatite
nanofiber composite as bone graft materials
Kalambettu Aravind and Dharmalingam Sangeetha*
Department of Mechanical Engineering, Anna University,
Sardar Patel Road, Chennai 600 025, Tamil Nadu, India.
*E-mail [email protected],
ABSTRACT
Nanofiber mats of polyether sulfone/nano hydroxyapatite (PES/nHA) were
prepared by electrospinning technique. The fabricated composites were
characterized using FTIR, XRD and SEM. The composite nanofiber mats were
subjected to in vitro biological studies namely bioactivity, haemocompatibility
and cytocompatibility. The XRD analysis showed an increase in the amorphous
nature of the composite with the addition of nHA (filler) suggesting an interaction
between the polymer matrix and the filler. The bioactivity studies performed
using Simulated Body Fluid (SBF) showed that the bioactivity was observed
to be higher in the composites containing nHA. It was observed that the
amount of protein (Bovine Serum Albumin) adsorption as well as the blood
coagulation decreased with the addition of the filler content which improved in
turn the cell adhesion and proliferation of osteoblast (MG-63) cells. The in vivo
studies, performed on a pilot scale by implantation of the PES/nHA in the tibia of
rabbits, suggested an intense inflammatory host response. Hence from the study,
it was concluded that although PES/nHA showed promising in vitro properties,
their unfavuorable in vivo response meant that these composites need to be
analysed further to determine the reason behind that inflammatory response and
the means of overcoming it.
1. INTRODUCTION
Total hip arthroplasty (THA) is a
commonplace
procedure,
though
for
younger patients there is a need to develop
new materials that can extend the life of the
artificial joint beyond 20 years. Dramatic
advances in the field of cell and molecular
biology, genetics, tissue engineering and
material science have given rise to the
remarkable new cross disciplinary field of
tissue engineering which uses synthetic or
naturally derived, engineered biomaterials
to replace damaged or defective tissues such
as bone, skin, and even organs. A potential
material for use as a scaffold in tissue
engineering must fulfill a number of
necessities
including
biocompatibility,
cytocompatibility and biodegradation to
nontoxic products within the time frame
required for the application, processability
to complicated shapes with appropriate
porosity, ability to support cell growth and
KEY WORDS:
Nano fiber composite;
Haemocompatibility;
Osteoblast;
Bioactivity;
In vivo.
proliferation,
along
with
appropriate
mechanical properties, as well as maintaining
mechanical strength during most part of the
tissue regeneration process [1]. When
developing new biomaterials for bone
regeneration, surface properties must be
modulated, ideally in order to mimic the
tissue to be replaced. Furthermore, a strong
bonding between the host bone and the
osteoconductive surface of the implant is
required [2]. Among the several methods
available for designing materials for tissue
engineering,
electrospun
fibers
have
garnered special interest over the recent
years in different areas of research and more
particularly, in the field of tissue engineering
as suitable materials for wound dressings,
tissue scaffolds and drug-delivery systems
[3-6].
Nanofiber composite, obtained by the
electrospinning process, offers a large
surface area to weight ratio which would
607 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619
help in the migration of cells as well as aid
cell growth. The polymer nanofiber
composites
could
be
made
from
biocompatible and biodegradable polymers
which could have potential applications in the
replacement of structurally or physiologically
deficient tissues and organs in humans. The
use of nanofibers in tissue restoration is
expected to result in an efficient and rapid
recovery process of the organ owing to the
large surface area offered by nanofibers
made from polymer. Polymeric nanofibers
have been successfully used for the
epithelialization of implants and the
construction of biocompatible prostheses,
cosmetics, face masks, bone substitutes,
artificial blood vessels, valves and drug
delivery applications [7]. Most commonly
used devices have a bearing surface of ultrahigh
molecular
weight
polyethylene
(UHMWPE) which has an unsolved problem
such as a lack of cytocompatibility properties
for promoting bone cell growth [8]. Sulfur
containing aromatic polymers have good
chemical stability and have been used in
medicine for various applications like
hemodialysis membranes, scaffolds for bio
mineralization, load bearing implants for
fixation in bone, etc [9-12]. The presence of
sulfur creates a stable bond between the
polymer and nano hydroxyapatite [9, 10].
This
holds
many advantages
over
conventional
UHMWPE/hydroxyapatite
composites where leaching of the apatite
and polymer particle are a matter of concern.
Contact of blood with artificial surfaces
during extracorporeal circulation procedures
is associated with activation of blood cells as
well as plasma proteolytic enzyme systems,
such as the complement, coagulation,
fibrinolytic
and
FXII–kallikrein–kinin
cascades. Numerous attempts have been
made to solve these problems by modifying
the surface chemistry of blood contacting
materials in order to make them more
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thrombo resistant. High haemocompatibility
is essential for medical devices such as
catheters and cardiovascular implants that
are in contact with blood in clinical use. At
present, one of the most serious challenges
is surface-induced thrombus formation
immediately after implantation of such a
device within the living system. Much
research has been focused on this problem
using various polymer materials or surface
modification methods [13]. Cell adhesion is
the most important aspect of cell interaction
with a biomaterial because it is the
prerequisite for further cellular activity
such
as
spreading, proliferation and
differentiation. Initial osteoblast material
interactions
may
be
conveniently
characterized by four stages: (i) protein
adsorption to the surface, (ii) contact of
rounded cells, (iii) attachment of cells to the
substrate, and (iv) spreading of cells. Initial
cell attachment is generally influenced by the
original surface characteristics of the
materials [7]. Hydrophobic properties of the
polymer are not favorable to direct cellular
adhesion and further population of cells [1415]. The introduction of bioceramics such as
nHA within the PES nanofiber is considered
to improve the hydrophilicity and cellular
affinity and thus to better allow its use as
tissue regenerative matrices and to promote
adhesions and proliferation of osteoblast
(bone-forming) cells. Hydroxyapatite (HA),
has been extensively investigated due to its
excellent biocompatibility, bioactivity and
osteoconductivity as well as its similarities
to the main mineral component of bone.
However, the poor compressive strength and
fatigue failure limits its applicability to the
low or non-load bearing sites in human body
[16-17].
Amongst the various polymers employed for
medical applications, polyethersulfone (PES)
has been used for separation and filtration
purposes. However, one of the drawbacks of
608 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619
the PES membrane is its low blood
compatibility which results in the adsorption
of proteins in the blood onto the PES
membrane surface and thus forming a protein
layer [18]. Prihandana et al [18] demonstrated
the improved the antithrobigenicity of PES
by coating it with fluoridated diamond like
carbon films. Ardeshirylajimi et al [19]
fabricated PES nanofiber scaffolds and
studied the differentiation of pluripotent stem
cells into osteoblastic lineage.
The present study i s focused on the
preparation of nanofibers of sulfur
containing aromatic polymer, PES and its
composite using electrospinning having a
bioceramic, nHA as the filler. The prepared
nanofiber composites were characterized
using X-ray diffraction (XRD), Fourier
transform infrared spectroscopy (FTIR), and
scanning electron microscopy (SEM). The
nanofiber composites were then subjected to
in vitro studies to evaluate their
bioactivity,
protein
adsorption,
haemocompatibility and cytocompatibility
properties inorder to test its sustability for
tissue engineering application. Subsequently
there in vivo performance was evaluated by
implanting them in the tibia of rabbits.
2. EXPERIMENTAL
2.1 MATERIALS AND METHODS
PES (Ultrason E6020P) and HA nano
powder (CAS 12167-74-7) were procured
from BASF, Germany and Sigma Aldrich,
USA respectively. Dimethyl formamide
(DMF) was used as a solvent, and was
obtained from SRL Pvt. Ltd, India. The
composition of the dope solution used for
this study was 2/0.5/7.5 of PES/nHA/DMF.
The dope solution was
dissolving PES in DMF and
was incorporated into it
quantities. The whole content
prepared by
the filler nHA
in calculated
was kept under
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magnetic stirring overnight and then
subjected to ultrasonication for 30 min prior
to the start of electrospinning process in
order to disperse the nHA uniformly in the
solution. In a typical electrospinning process,
t h e p olymer solution with a specific
concentration of nHA was loaded onto a 2
ml syringe which was linked to a high
voltage power supply with a capacity to
generate high voltage of up to 50 kV. The
flow rate of the syringe pump was regulated
using the PICO Espin 2.0 version software.
The electrospinning was performed with an
electric voltage supplied at 20 kV at a needle
tip to collector distance of 15 cm. The flow
rate was adjusted to 0.3 ml/h and the
collecting drum was regulated to rotate at a
speed of 2000 rpm.
From amongst the prepared PES/nHA
nanofiber
composite
with
different
concentrations of nHA, the PES nanofiber
composite with 5 wt% of nHA showed the
best fiber formation. Hence PES/nHA
nanofiber composite mat with 5 wt% of nHA
alone was considered for further studies.
2.2 Characterization Studies
2.2.1 XRD
The phase analysis of the nanofiber
composite samples were done by XRD using
35 mA, and 40 kV current, with a
monochromatic
CuKα
radiation
(λ=1.5405˚A) with a step size of 0.04 º 2θ,
a scan rate of 0.02º 2θ/s, and a scan range
from 2θ = 10 to 60°.
2.2.2 FTIR
The functional groups present in the
polymer and the interaction between the
polymer/nHA nanofiber composites were
analyzed by using Alpha T Bruker Optics
FTIR spectrophotometer.
The spectra
obtained were recorded in transmission mode
within the scanning range of 4000–500cm-1.
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2.2.3 SEM
2.5 Blood coagulation
The morphology and dispersion of particles
in the polymer matrix were observed using
HITACHI S-3400 model SEM. The surface
of the materials was sputter coated with gold
before being subjected to SEM in order to
make them electro conductive.
Blood coagulation tests were carried out
with PES and its nanofiber composite
individually using blood samples collected
from healthy individuals. Buffered citrate
was added to the blood samples which
functioned as an anticoagulant agent. The
samples were separately immersed in the
blood contained in the test tubes and left
undisturbed for 2 h. The morphology of the
cells before and after the treatment with the
samples was analyzed using Light
microscope (Labomed, LX-400).
2.3 Bioactivity
Simulated body fluid (SBF) was prepared in
laboratory according to procedure developed
by Kokubo [2, 9]. In vitro tests were
carried out to assess the bioactivity of
the
prepared composite samples. The
samples were immersed in SBF solution for
30 days and maintained at 37 ºC. The
samples were retrieved from SBF solution,
dried and then their surface was analyzed by
SEM
to
observe
the
growth
of
hydroxyapatite.
2.4 Protein adsorption
The protein adsorption experiments were
made with Bovine Serum Albumin (BSA)
solutions. The known concentration of BSA
was prepared in phosphate buffered saline
(PBS, pH=7.4). The nanofiber composite
with an area of 1 cm2 was incubated in
distilled water for 24 h, washed 3 times with
PBS solution, and then immersed in the
protein solution for 2 h.
After protein adsorption, the membranes
were carefully rinsed 3 times with PBS
solution and then rinsed with distilled water.
The adsorbed proteins were quantitatively
eluted with 1.0 ml of 2% SDS solution for 6
h. The amount of protein in the sodium
dodecyl sulfate (SDS) solution was
quantified by protein analysis (Micro BCA
protein assay reagent kit) [20].
2.6 MTT Assay
Cytotoxicity studies using the nanofiber
composite were analyzed in 96 well plates
using normal and osteoblast cell lines (MG63) by MTT assay. MG-63 cell lines were
cultured using Dulbecco’s modified Eagle’s
medium (Himedia) supplemented with 5%
fetal bovine serum (FBS) and 1% penicillinstreptomycin and then seeded into the 96
well plate. The wells were sterilized with
70% ethanol followed by UV treatment
for 4 h and were neutralized with
phosphate buffer (pH 7). The wells without
the polymer samples were the control groups
for the experiment. The MG-63 cell lines
were seeded at a density of 6-7x103 cells per
well and incubated at 37 °C in a humidified
atmosphere containing 5% CO2. In all
culture conditions, the medium was renewed
every 24 h. After 3 days of incubation, the
supernatant of each well was removed and
washed with PBS. MTT, diluted in serumfree medium, was added to each well and
the plates incubated at 37 °C for 3 h. After
aspirating the MTT solution, acidified
isopropanol (0.04N HCl in isopropanol) was
added to each well and pipetted up and
down to dissolve the dark blue formazan
crystals and then left at room temperature
for a few minutes to ensure the dissolution
610 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619
of all crystals. Finally, the absorbance was
measured at 570 nm using an ELISA
reader. Each experiment was performed at
least three times for reproducibility.
2.7 In vivo studies
PES/nHA (5 wt%) were implanted in the tibia
of rabbits and the type of host response was
studied histologically. Institutional Animal
Ethical
Committee
(IAEC)
approval
(Approval number: IAEC/ XXX/SRU/
225/2012) was obtained at Sri Ramachandra
University, Chennai, prior to performing the
in vivo experiments. Three healthy male New
Zealand white rabbits, aged about 16 weeks
and weighing between 1.8 and 2 kg were
selected for the study.
The PES/nHA
composite samples were autoclaved prior to
implantation.
2.7.1 Preparation of the animals
The medial side of rabbit tibial proximal
epiphysis was chosen as the site of
implantation since it had the least muscle
attachments and was considered to be
anatomically favourable. For this purpose, the
fur around the proposed site of surgery was
removed and the site cleaned using povidoneiodine solution.
2.7.2 Anaesthesia protocol
The three rabbits were anaesthetised using a
combination of diazepam (5 mg/kg) and
ketamine (60 mg/kg) i.m. for induction and
maintained using Isoflurane until the end of
the surgical procedure.
2.7.3 Surgical procedure
Once the rabbits were sufficiently
anaesthetised, the surgical site was exposed
by placing an incision on the skin at the
medial side of the tibia and deflecting the
underlying fascia. A carbide fissure bur fixed
to a dental handpiece was used to drill two
holes in the bone with each hole measuring
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about 5 mm wide and depth until the marrow
was reached. The two holes were placed in a
straight line and 1 cm apart. The PES/nHA
composite samples were cut into tiny pieces
(approximately square shaped of about 1
mm2) using a pair of sterile scissors. The
graft material was then slightly wet with
saline and then implanted into the first defect
in as much quantity so as to slightly overfill
the first defect. In the second defect of all the
three rabbits, a biphasic calcium phosphate
commercial bone graft material (DM bone,
MetaBiomed,
Korea)
was
filled.
Subsequently, the soft tissues overlying the
bone were sutured using catgut sutures and
the skin was sutured using silk suture threads.
The surgical site was then protected by a
bandage.
The animals were then revived and kept
under observation. Analgesia was ensured by
administering Ketorolac (Ketorol, Dr Reddy
Laboratories, India) injections i.m. for 10
days. Antibiotic coverage was provided for 7
days post operatively through cefalexin (15
mg/kg SC). The animals were then
maintained for 8 weeks and then euthanised
by giving an overdose of ketamine i.v.
Subsequently, the right tibia of each animal
was harvested and fixed in 10% neutral
buffered formalin, decalcified in 10% formic
acid, dehydrated in series of graded alcohol
and embedded in paraffin. From these
samples, 3 – 4 µm thick vertical serial slices
were prepared using a microtome and surface
staining was performed with haematoxylin
and eosin (H&E). Some of the samples were
not H&E stained and used for SEM analysis
to study the cell spreading. The H & E
stained bone sections were examined under
Optika Trinocular fluorescence microscope
and evaluated for the following parameters
1. Detection of inflammatory cells
2. Detection of type of healing
3. Detection of osteoblasts/ osteoclasts both
around as well as on the surface of implant.
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3. RESU
ULTS AND DISCUSSIION
3.1 XRD
D
The XRD
D is a verssatile techniique to stud
dy
the grow
wth of any crystalline
c
phase on oth
her
amorphous or crysttalline matrrix. Hence in
the preseent study, the
t capabilitty of PES to
facilitate the grow
wth of HA on it was
w
X
The XRD patteern
investigaated using XRD.
of electrrospun PES
S nanofiber is shown in
Figure 1a,
1 which co
onfirmed th
he amorphou
us
nature of PES. The XRD
D pattern of
electrosp
pun PES placced in SBF for 30 days is
shown in
n Figure 1b
b. From the Figure, th
he
evidence for the gro
owth of HA was noted by
b
the appeearance of a peak closee to 32° (2θθ).
Hence th
he capability
y of PES to permit
p
grow
wth
of HA on
o its surfaace was verrified. As th
he
intensity of the refllection due to HA was
w
found to be very low
w, the grow
wth of HA on
o
PES/nHA
A nanofibeer compositte was also
studied using the same SBF. The XR
RD
patterns of PES/nH
HA nanofib
ber composiite
and of th
he same mateerial placed in SBF for 30
3
days are represeented in Figgure 1c annd 1d
respecctively. Preesence of H
HA phase was
evidennt in PES/nnHA compossite nanofibeer by
the chharacteristic reflections at 26°, 32°,, 40°,
47°, 550°, 53° andd 64° (2θ). IIn Figure 1dd, the
intenssity of thee reflectionns due to HA
increaased, thus prroving the ggrowth of H
HA on
the pprevious nnHA already presentt in
PES/nnHA compoosite nanofi
fiber. Hencee the
study confirmed tthat the inheerent properrty of
F was
nHA ttiny crystalss to grow furrther in SBF
not aaffected byy PES. T
Though PES
S is
hydropphobic, thee growth oof nHA att the
interfaace was not suppresssed. This sstudy
confirrmed that either PES
S or PES//nHA
nanofi
fiber compossite could bee used as a m
matrix
for thee growth of nHA in SBF
F. (Fig. 1)
3.2 FT
TIR
The fformation oof apatite layer over the
nanofi
fiber was cconfirmed by ATR-F
FTIR
spectrroscopy. A
ATR-FTIR sspectra of PES
nanofi
fiber and its nanofiberr compositee are
shownn in Figure 2. All thhe spectra sshow
615 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619
their strongest band in the spectrum region
below 1800 cm-1. The peaks observed at
1125 and 1242 cm-1 correspond to diaryl
sulfone (Ar-SO2-Ar) and aryl ether (Ar-OAr) groups respectively, which is the back
bone of PES [21]. The vibration of aromatic
C=C group that occur at 1586 cm-1 belong
to benzene ring. The broad band at about
2800 cm-1 correspond to the absorbed
hydrate and the sharp medium and short
peaks at 632 and 3570 cm-1 corresponds to
stretching vibrations of lattice OH- ions of
hydroxyapatite. And also the peaks at 632
and 3570 cm-1 are the characteristic bands
for stoichiometric nHA. The symmetric P-O
(PO4-3 ion) stretching mode for nHA occurs
at 995 cm-1 which indicates typical nHA
structure (Figure 2d). From the investigation,
it is proved that the nanofiber composite
soaked in SBF after nHA addition are able to
induce apatite nucleation. (Fig. 2)
3.3 SEM
The morphology of PES and its nanofibers
composite are shown in the SEM image in
Figure 3 (a, b). The average diameter of the
obtained fiber between 150-480 nm offers
more surface area to weight ratio that helps
the deposition and growth of apatite and
provides huge surface area for the
proliferation
of
bone forming cells
(Osteoblast) and also improves the
angiogenesis for blood vessels to penetrate
through the nanofiber [22]. Figure 3b shows
the PES nanofiber with adhereded nHA that
acted as stimuli for the growth of apatite
when immersed in SBF. (Fig. 3)
3.4 Bioactivity
The bioactivity of the nanofiber mats of
polymer and the composite was observed
from the SEM images (Figure 4), for
evidence of apatite formation after 30 days
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of immersion in a metastable calcium
phosphate solution (SBF). From the earlier
studies [23] it was proved that the formation
of apatite layer on polymer/nHA composite
was due to the presence of nHA which was
used as a stimulus and has the ability to
induce the formation of a bone-like apatite
layer over the nanofiber. Hence in the
present study also, nHA was used to induce
bioactivity. Figure 4b showed that the nHA,
present in the nanofiber composite, acted as a
nucleation site resulting in increased apatite
formation over a period of time and is
expected to not only promote the tissue
growth adjacent to the implant site but also
facilitate a strong bond between the tissue
and the implant. It was observed that the
PES nanofiber, after 30 days of immersion
in SBF, showed evidence of apatite
formation and from which it was inferred
that, the minerals present in the SBF
encouraged apatite formation albeit at very
low level as shown in Figure 4a. Our results
were in agreement with previous reports
where nHA was used as a stimulus for
apatite formation [24]. (Fig. 4)
3.5 Protein adsorption
Protein adsorption on the material surface is
a common phenomenon during thrombus
formation upon contact of the biomaterial
with blood. Thus, the amount of protein
adsorbed on the
PES
nanofiber
is
considered to be one of the important
factors
in
evaluating
the
haemocompatibility [20, 21]. Figure 5
shows adsorption of BSA on the
nanofiber and its composite. From these
observations it was found that the amount of
protein adsorbed on the surface of apatite
forming nanofiber composites was lower
(Figure 5b and 5d) than that of the plain
nanofiber (Figure 5a and 5c).
Hydrophobicity of the polymer favors the
adsorption of protein on the surface and may
616 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619
lead to undesirable results, such as platelet
adhesion, aggregation and coagulation [25].
The incorporation of nHA increased the
hydrophilicity of the nanofiber mat thereby
decreasing the protein adsorption, which was
expected to improve its biocompatibility.
(Fig.5)
3.6 Blood coagulation
From the microscopic image in 6a, the
floating of blood cells with spherical shape
was observed which was due to the presence
of anti-thrombogenic agent (buffered citrate)
and is seen prior to contact with the nanofiber.
The morphology of blood cells changed along
with the formation of a clot when the
polymer nanofiber c a m e in contact with
blood. The microscopic image in figure 6b
was taken after 2 h contact with PES
nanofiber. This observation underlined the
thrombogenic nature of bare PES nanofiber.
Interestingly
diminishing
of
blood
coagulation with oval shaped blood cells
were observed on apatite formed nanofiber
composite (Figure 6c).
The observed decrease in thrombogenicity
could be attributed to the enhanced
hydrophilicity of the nanofiber [26], which in
turn resulted in decreasing adsorption of
fibrinogen, which eventually lowered the clot
formation [27]. The results revealed the fact
that the formation of blood clot constantly
decreased with addition of nHA in the feed
mixture. The results may be explained on the
basis of the fact that nHA increases
hydrophilicity of the polymer and therefore,
are not expected to induce any damage to
blood cells or any change in the structure of
the plasma proteins [28]. Hence, based on the
above studies, it was demonstrated that the
antithrombogenic property of the PES
nanofiber was achieved with apatite formation
and improved the haemocompatibility of the
nanofiber. (Fig. 6)
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3.7 Cell Morphology
The morphology of osteoblast cells (MG-63)
on the nanofiber composite was observed in
vitro by SEM images after 5 and 10 days of
seeding. It was observed that the bare
nanofiber mat remained unchanged after
seeding with bone forming cells as seen from
the SEM images shown in figures 7a and 7b.
SEM images in figures 7c and 7d shows the
migration of osteoblast cell lines over the
PES/nHA
nanofiber
composites
after
incubation in SBF. It was noted that the
addition of nHA increased the hydrophilicity
of the scaffold and thus eventually helping the
adhered osteoblastic cells to spread and
migrate on the surface of the nanofiber
composite. It has been reported that the
osteoblast-like cells prefer more hydrophilic
surfaces [29]. The SEM examination of the
osteoblast cell (MG-63) growth on the
surface of the apatite formed nanofiber
composite showed that almost the whole
surface of the nanofiber composite was
covered with the cells in contrast to the case
with the bare PES nanofiber. This implied
that the addition of nHA promoted better cell
adherence and proliferation. (Fig. 7)
3.8 Cell viability
The viability of cells in terms of percentage,
after 40 and 80 h of seeding on the nanofiber
composites are shown in Figure 8. From the
figure, it was evident that after 80 h, 65-90%
of cells could survive on these nanofibers.
Not surprisingly, the number of cells on
the apatite formed polymer nanofiber
composite was noted to be always higher
than that on the plain polymer nanofiber
during the culture period and was likely due
to the higher cell adhesion on the apatite
formed nanofiber [30]. It was also evident
that, the viability of cells on the bare PES
nanofiber decreased with culturing time.
From the observed result, it was deciphered
617 Dharmalingam Sangeetha J.Biosci Tech,Vol 6(1),2015,607-619
that the apatite formation on the PES/nHA
apparently increases the cytocompatibility.
(Fig. 8)
3.9 In vivo study
The results of the in vivo studies using
PES/nHA composites were disappointing. All
the three samples showed negligible evidence
for new bone formation as visualized in
figure 9. The histology pictures showed a
strong presence of inflammatory cells and
multinucleated giant cells. There was also
evidence for local bone necrosis surrounding
the implant site. This unfavourable host
response was observed despite the favourable
in vitro response with MG 63 cell line. This
contrary observations could be explained by
considering that while in in vitro studies, the
scaffolds are exposed to only one cell line
(MG 63 in the present case), in the in vivo
experiments, the entire host immune system
(including macrophages, inflammatory cells
and interleukins) comes into play.( Fig. 9)
4. CONCLUSION
The characterization of the electrospun
PES/nHA nanofiber composite using XRD
and FTIR discovered the apatite formation.
Hydrophilicity of the composite enhanced
by the nHA was identified with low
adsorption of protein which leads to
improved cell viability of the biomaterial.
The PES/nHA composite nanofiber showed
excellent haemocompatibility compared with
bare PES nanofiber due to low protein
adsorption
rendered
by
improved
hydrophilicity of the polymer. In vitro study
combined with SEM characterization of
apatite formed nanofiber revealed substantial
biomineralization.
The
exhibited
hydrophilicity of the nanofiber composite
appeared to have enhanced cell adhesion and
proliferation rates of osteoblast (MG-63) cell
ISSN: 0976-0172
Journal of Bioscience And Technology
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lines which
were further confirmed by
SEM. In addition, the in vitro cell viability
of the osteoblast also increased the life span
of the biomaterial. Surprisingly, the success
achieved in the in vitro studies was not
reproducible in the in vivo studies. But
considering the favourable in vitro cell
response of MG 63 cells, it would be justified
to attempt either PEGylation or other
chemical
modifications
(such
as
grafting/Layer By Layer technique) of PES
and then evaluate the in vivo response.
However, the best method would be to find
out first the reason behind the adverse in vivo
response and later attempt at overcoming the
limitation. It is widely recognized that by
increasing the hydrophilicity of PES surface
better haemocompatibility can be achieved.
At the same time, the roughness of the PES
scaffold is also known to determine the cell
response. One method of modifying the
surface roughness is through CO2 pulsed
layer irradiation [31].
Acknowledgements: The authors would like
to thank Indian Council of Medical
Research (ICMR), New Delhi, India for
funding the study (Vide letter No.
5/20/5(Bio)/09-NCD letter dated 26.02.2010)
and All India Council for Technical Education
(AICTE), New Delhi, India for the Doctoral
fellowship awarded to Aravind (vide their
letter no. vide their letter no. 110/RID/NDFPG (5)/2009-10).
The help
rendered by SRMC, Chennai in carrying out
the study is also gratefully acknowledged.
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