journal of the mechanical behavior of biomedical materials 60 (2016) 243–255

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Research Paper

Enhanced bone regeneration with carbon nanotube

reinforced hydroxyapatite in animal model

Susmita Mukherjeea, Samit Kumar Nandib,n, Biswanath Kunduc,nn,

Abhijit Chandad, Swarnendu Send, Pradip Kumar Dase
a
School of Materials Science and Nanotechnology, Jadavpur University, Kolkata 700032, India
b
Department of Veterinary Surgery and Radiology, West Bengal University of Animal and Fishery Sciences,
Kolkata 700037, India
c
Bioceramics and Coating Division, CSIR-Central Glass and Ceramic Research Institute, Kolkata 700032, India
d
Department of Mechanical Engineering, Jadavpur University, Kolkata 700032, India
e
Department of Veterinary Physiology, West Bengal University of Animal and Fishery Sciences, Kolkata 700037, India

art i cle i nfo ab st rac t

Article history:
Received 11 November 2015
Received in revised form
1 February 2016
Accepted 3 February 2016
Available online 10 February 2016
In order to improve the inherently poor mechanical properties of hydroxyapatite (HAp) and
to increase its feasibility as load bearing implant material, in the present investigation,
functionalised (HFC1 and HFC2) and non-functionalized (HC1 and HC2) multi-walled carbon
nanotubes were used as reinforcing material with HAp. Significant improvement with
respect to fracture toughness, flexural strength and impact strength of the composites was
noticed. In vitro biological properties of HAp–carbon nanotube (CNT) biocomposites have

Keywords: also favored uniform and systematic apatite growth on their surface. Subsequently, in vivo

CNT-HAp composite osseous ingrowth at bone defect of rabbit femur was evaluated and compared using

functionalized CNT radiology, push out test, fluorochrome labeling, histology and scanning electron micro-

In vivo scopy after 2 and 4 months respectively. The results demonstrated growth of web like soft

radiology callus from the host bone towards the implant, ensuring strong host bone interaction.

push out test Toxicological studies of the liver and kidney cells exhibited no abnormality, thereby

fluorochrome labeling confirming non-toxicity of the CNT in the animal body. Host–implant biomechanical

histology strength showed high interfacial strength of the composites, indicating their high

scanning electron microscopy potentials to be used for bone remodeling applications.

& 2016 Elsevier Ltd. All rights reserved.

1. Introduction (Johnell, 1997). Complication and scarcity issues associated

with allografts and autografts created a huge demand for
Orthopedic surgery due to recurrence of different bone dis- synthetic biomaterials. Of the different forms of synthetic
orders is expected to reach 6.6 million per annum by 2020 biomaterials, calcium phosphate ceramics, particularly

n
Corresponding author. Tel.: þ91 9433111065.
nn
Corresponding author. Tel.: þ91 9831772081.
E-mail addresses: samitnandi1967@gmail.com (S.K. Nandi), biswa_kundu@rediffmail.com (B. Kundu).

http://dx.doi.org/10.1016/j.jmbbm.2016.02.005
1751-6161/& 2016 Elsevier Ltd. All rights reserved.
244 journal of the mechanical behavior of biomedical materials 60 (2016) 243 –255
hydroxyapatite [Ca10(PO4)6(OH)2] or HAp has been the ideal
choice as a bone-substitute material since it constitutes the
main mineral phase of bone for its excellent biocompatibility
and osteoconductive property. Inspite of its superb bone-
bonding property, its poor mechanical properties, especially
its low strength and toughness impede its application as a
major load bearing implant material (White et al., 2007).
Instead, it is chiefly used as a coating material or scaffold
for tissue engineering. To enhance the mechanical properties
of the brittle ceramic matrix, without sacrificing its biocom-
patibility, different dopants like zinc, magnesium, silver
(Webster et al., 2004) or secondary phases like zirconia,
alumina, titania, carbon fibers, carbon nanotubes etc. have
been widely used as reinforcing materials (Kong et al., 1999;
Lopes et al., 1999; Park and Vasilos, 1997; Shen et al., 2001;
Zhang et al., 1997).
Carbon nanotubes (CNTs) are getting considerable atten-
tion in recent past owing to their good mechanical properties
together with high tensile strength, high resilience, flexibility
and other unique structural and physicochemical properties
(Iijima, 1991; Khabashesku et al., 2005; Sambarkar et al., 2012).
CNTs have widely applied as a reinforcement material to
improve the strength and toughness of the HAp matrix (Kaya,
2008; Najafi et al., 2009). Unique structural dimensions of the
carbon nanotubes (diameter of 0.5–2 nm, density of 0.2–0.7 g/
cm3 and aspect ratio of 103–104) result in its superb efficacy as
a reinforcing agent (Dresselhaus et al., 2004; Iijima, 1991;
Ruoff and Lorents, 1995). In addition, for their strong inter-
chain van der Waals interactions, CNTs are very inert and do
not disperse in any common solvents. Its poor dissolution
property restricts the applications in many situations (Balazsi
et al., 2003; Baviskar et al., 2012; Lupo et al., 2004; Rul et al.,
2004; Xia et al., 2004). Further dispersability and thereby their
reactivity, carboxylation of the nanotubes chains were
enhanced using strong acids (Eitan et al., 2003; Hirsch, 2002;
Sun et al., 2002).
Our aim was to develop a strong HAp based implant
material that may be used for bulk applications. The present
study mainly focused on non-articulating bones that suffer
low wear but bear a high load. In our previous publications,
we synthesized and studied detailed physico-mechanical
properties of different HAp–CNT composites, using both
unreacted chains as well as the functionalised chains
(Mukherjee et al., 2015). Salient observations are given in
the Supplementary material (SM) section. Nonetheless, we
have established through in vitro studies that clearly showed
the composites offered advantage on mechanical properties
over that of monophasic HAp ceramics, especially in terms of
fracture toughness and flexural strength. Also the bioactivity
study of the samples, when immersed in simulated body fluid
for a prolonged time, proved the materials to be highly
favorable as a bone implant material. In this work, a detailed
in vivo pre-clinical study of the HAp–CNT composite was
performed on healthy adult rabbits using bone defect model
to investigate the in vivo response of HAp–CNT biocomposites
and to study their interfacial interaction as well the biome-
chanical strength offered at the bone–implant interface.
Chemical and physical nature of the composite developed
in a nutshell is given in SM Fig. A.
2. Materials and methods
2.1. Preparation of HAp–CNT composite
The multiwalled carbon nanotubes (MWNT) used in the study
were procured from Nanoshel LLC, USA while the hydroxya-
patite was synthesized in the laboratory, using analytical
grade ortho-phosphoric acid and calcium hydroxide (S.D.
Fine-Chem Ltd., India) by the wet chemical method.
To facilitate uniform dispersion, the carbon nanotubes
were functionalised using strong acid solutions (3:1 mixture
of concentrated sulfuric and nitric acid), which were recon-
firmed through FTIR studies as well as optical images of the
aqueous dispersion. The HAp–CNTs biocomposites were pre-
pared using both the treated and the untreated nanotube
chains following established protocols in our laboratory
(Mukherjee et al., 2015, 2014). In a nutshell, the calcined
HAp powders were ball milled with MWNT (both functiona-
lised and non-functionalised) in a high energy planetary ball
mill using acetone medium. The milled powders were sieved,
green compacts pressed uniaxially and finally sintered under
argon atmosphere (two-step sintering done: first upto 700 1C
at a heating rate of 3 1C/min and then upto 1250 1C at a
heating rate of 6 1C/min). Two different concentrations were
studied: 1% by weight of MWNT and 2% by weight of MWNT,
for each of non-functionalised and functionalised types that
were named as: HC1: HApþ1%, MWNT; HC2: HApþ2% MWNT;
HFC1: HApþ1%, f-MWNT and HFC2: HApþ2% f-MWNT.
2.2. Physico-chemical, mechanical and in vitro
characterization of HAp–CNT composites
The phase purity of the biocomposites were analyzed using X-
Ray diffraction (XRD, Ultima III Rigaku diffractometer) technique
using CuKα radiation (λ¼ 0.154 nm) while the chemical structure
was analyzed through fourier transformed infrared spectroscopy
(FTIR, IR Prestige 21, 200VCE, Schimadzu) in mid IR region (i.e,
4000–400 cm  1). The microstructure and morphology of the
composites were studied by field emission scanning electron
microscopy (FESEM, Hitachi, S 4800) and high resolution trans-
mission electron microscopy (HRTEM, JEOL, JEM2100). Open
porosity and bulk density of sintered composites were estimated
by Archimedes' water displacement method using formulae
[bulk density¼D/(W S) g/c.c. and open porosity¼ (WD)/
(WS)  100%], where D, W and S are dry, soaked and suspended
weight respectively; relative density was calculated from theore-
tical density. The mechanical properties of the composites were
evaluated by studying the hardness, fracture toughness, flexural
and impact strength of the samples. The in vitro biological
studies of the composites were tested by performing their
biocompatibility study and bioactivity study in simulated body
fluid in our previous publications (Mukherjee et al., 2015, 2014).
Hardness and fracture toughness of the sintered compo-
sites were measured by Vickers hardness tester (LECO, LV-
700AT) using indentation method (ASTM C1327)(ASTM-
C1327-15, 2015). Hardness values were calculated using the
formula: HV¼1.8544*(P/d2), where HV is Vickers' hardness, P
is applied load and d is average diagonal length for an
individual indent. Fracture toughness was calculated using
 journal of the mechanical behavior of biomedical materials 60 (2016) 243 –255
the relation: KC ¼δ(E/H)1/2  (P/c3/2), where E is the Young's
modulus, H is the hardness and δ is constant depend on
geometry of indenter (0.01670.004 for standard Vickers dia-
mond pyramid indenter). Flexural strength was measured
using Universal Testing Machine (Instron UTM 4204) by 3-
point bend test with rectangular specimens and using the
equation: sf ¼(3Fl)/(2bd2), where F is load at fracture failure, l
is span length, b and d are breadth and thickness of samples.
Finally, impact strength of the samples was evaluated using
falling weight test method (slightly modifying the ASTM
D5628)(ASTM-D5628-10, 2010) to estimate the impact resis-
tance of the composites.
2.3. In vivo study
Here, the in vivo experiments were carried out on healthy
adult New Zealand rabbits, by creating bony defects at the
distal femur of the animals and then introducing the carbon
nanotubes reinforced bioceramic implants. Different steps of
the implantation surgical procedure are pictorially given in
SM Fig. B. The whole work was carried out at the West Bengal
University of Animal and Fishery Sciences, Kolkata, India as
per the guidelines of the Institutional Animal Ethics Commit-
tee of WBUAFS (Permit no. E.C.765 dated 13.12.2012). A total of
twenty (20) adult New Zealand rabbits of either sex and
average body weight of 1–1.5 kg were used for the study.
The rabbits were randomly distributed into five groups with
4 animals in each group; Group 1: control, HAp; Group 2: HC1;
Group 3: HC2; Group 4: HFC1 and Group 5: HFC2. The implants,
prepared from the pre-sintered pellets, were cylindrical in
shape (diameter 2 mm  height 4.5 mm) and sintered at
1250 1C under argon atmosphere.
2.3.1. Surgical procedure
During the experiment, the animals were housed in separate
cages, maintaining strict hygiene along with comfortable
environment and a balanced standard diet. The implantation
surgery was carried out under standard aseptic conditions,
following the standard protocol. The animals were subjected
to general intramuscular anesthesia using xylazine hydro-
chloride (Xylaxins, Indian Immunologicals, India @1 mg/kg
bodyweight) and ketamine hydrochloride (Ketalars, Parke-
Davis, India). A small incision (about 3 cm long) was made on
the skin of the distal femur. The defect site (diameter
0.4 cm  length 0.3 cm) was prepared by drilling the cortical
bone with a micromotor drill while flushing with copious
amount of cold and sterile normal saline water to minimize
the mechanical and thermal trauma to the bone. After press
fitting the implants at the prepared sites, the muscle, the
subcutaneous tissue, and the skin were sutured in layers to
secure the implant in position (SM Fig. B).
Postoperatively, each animal was administered with cefo-
taxime sodium injection (Taxims, 50 mg IM twice daily;
Mapra India, India) and injectable meloxicam (Melonaxs,
0.1 mL once daily for 5 days; Intas Pharmaceuticals, India)
along with daily dressing and changes of the surgical wounds.
The operated animals were observed carefully upto 4 months
postoperatively, to note any associated signs of swelling, local
inflammatory reactions, lameness, fracture, repair or hema-
toma edema, by visual or manual examinations.
245
2.3.2. Local inflammatory reactions
The operated animals were observed carefully from day
0 upto 4 months postoperatively, to note any associated
signs of swelling, local inflammatory reactions, lameness,
fracture, repair or hematoma edema, by visual or manual
examination.
2.3.3. Radiological study
Sequential radiographs of the femur bone were recorded at
regular intervals (days 0, 1, 2, 3 and 4 month postoperatively)
in a medical diagnostic X ray machine (300 mA, M.E. X-Ray,
India) to study the status of the implant and the degree of
host bone–material interaction.
2.3.4. SEM and histological analysis
The radiological results were further verified by microscopic
evidences (SEM, JEOL, JSM, 5200 Model) as well as by histolo-
gical investigations of the operated sites in order to under-
stand the distribution and development of the osseous tissue
in-growth at the defect site. After sacrificing the animals at
the end of 2 and 4 month post-operatively, the bone speci-
mens were collected, washed with normal saline, cut into
thin sections containing the implant and preserved in 10%
formalin. For histological analysis, decalcified tissues (using
Goodling and Stewart's fluid) were stained with hematoxy-
lene and eosin to observe the matrix formation in an optical
microscope. For scanning electron microscopy (SEM) analysis,
the osseo-samples were fixed in 2% E.M. grade glutaraldehyde
phosphate solution for 48 h, washed twice for 30 min with
phosphate-buffered saline (pH 7.4) and distilled water, next
dehydrated in a series of graded alcohol solutions, followed
by final drying in 100% ethyl alcohol. Dried samples were
sputter coated with gold before imaging using a field emis-
sion scanning electron microscope (FESEM, LEO, UK).
2.3.5. Fluorochrome labeling study
Fluorochrome labeling analysis of the implanted bony sec-
tions was carried out using an Orthoplan microscope (Excita-
tion filter, BP-400 range, Leitz, USA) to understand the new
bone formation as well as the mineralization process. For this
study, oxytetracycline dehydrate (OTC) (Pfizer India, India), a
bone forming marker, was administered to each animal
(intramuscularly @25 mg/kg body weight) 25 days before the
sacrifice of 2 month samples i.e., on days 35, 36 and after a
gap of 6 days on day 43, 44 (2-6-2) post-operatively for double
toning of the new bone. Again on days 95 and 96 and then on
101, 102 days, OTC was administered to each animal for
4 month sample. For the analysis, thin and undecalcified
sections were prepared from the implanted bone specimens
which were observed under UV light. Data obtained was
further taken in Leica DM 2000 Bright light phase contrast
and fluorescence microscope including Leica qwain software.
In the said microscope, area of bone formation (golden yellow
fluorescence) has been measured in mm2 and subsequently
converted to percentage of bone formation and finally statis-
tical analysis was carried out.
2.3.6. Biomechanical study
To assess the mechanical strength offered by the implant at
the bone–implant interface, push-out test of the samples
246 journal of the mechanical behavior of biomedical materials 60 (2016) 243 –255
were performed at 2 and 4 month postoperatively. After
sacrificing the animals, the proximal and distal femur meta-
physes were excised, leaving the diaphyseal segment con-
taining the implants. The bones were divided longitudinally
with a band-saw to give access to the implant inside the
intramedullary canal. After removal of soft tissue, the bone
specimens were cut into thin sections, each section contain-
ing the implant and preserved in formalin. During the test,
resin mounted samples were placed on a metal base, contain-
ing a circular hole of diameter little larger than the implant-
perimeter (the test set-up is schematically shown in SM Fig.
C. After aligning the sample with the plunger axis, load was
applied with a plunger attached to the Instron (5500R UTM,
UK), until the failure load is attained. Three samples of each
group were used for the push out test. To calculate the
contact area for each specimen, the thickness of the cortical
bone around the implant was measured at four sites and
their mean was approximated as the actual cortical thick-
ness. The product of the height and circumference of the
implant dissected from the segment was measured as the
contact area between the implant and the surrounding bone.
In each case, the failure load was divided by the contact area
to calculate the bonding strength between bone and implant
(average value was recorded). Before each test, the machine
was calibrated and special care was taken to ensure the
linearity between the loading force and the long axis of each
implant.
2.3.7. Toxicological study
In order to evaluate toxicological effects of HAp–CNT compo-
sites, histology of two vital excretory organs, viz., liver and
kidney, where end products of all metabolisms get deposited,
was studied. After the sacrifice, the liver and the kidney of the
animals were collected and preserved in 10% buffered formalin
solution. The sections were processed and stained with hema-
toxylene and eosin solution for microscopic examination.
3. Results
3.1. Material characterizations
The composites' phase evolution characterized through XRD
and FTIR have been discussed in detail in our previous papers
(Mukherjee et al., 2015, 2014). In a nutshell, the results
suggested that the composites possess the basic structure
of HAp and the association of the HAp with the CNT chains is
purely physical, with no new bonds being observed in the
FTIR patterns. Main phase matches with pure HAp (JCPDS
PDF #09-0432) and both functionalized/non-functionalized
composites did not show any new compound peak as well.
In the FTIR, characteristic peaks of stretching vibrations of
phosphate group and hydroxyl group corresponded the HAp
structure (Anee et al., 2003; Rehman and Bonfield, 1997),
while the later group is absent for the composites, possibly
due to sintering. HFC1 and HFC2, on the other hand, had extra
carbonate band present (Rehman and Bonfield, 1997). Both
XRD and FTIR patterns are shown in SM Figs. D and E
respectively.
Table 1 – Variations of open porosity and bulk density of
HAp–CNT composites.
Sample Bulk Standard Open Standard Relative
density, deviation porosity, deviation density,a
g/c.c. % %
HAp 3 0.21 2.52 0.238 95.07
HC1 2.93 0.23 2.17 0.4 92.61
HC2 2.8 0.12 7.93 0.382 88.53
HFC1 2.92 0.12 3.49 0.603 92.47
HFC2 2.86 0.22 5.86 0.567 90.65
a
Theoretical density of HAp is considered as 3.167 g/c.c.
Open porosity and bulk density as function of CNT content
of the sintered specimens, measured by Archimedes' water
displacement principle, showed decrement tendency of bulk
density with increasing CNT content and the density was
found to be minimum for 2% loading and accordingly,
porosity was maximum for the same. Variations in open
porosity and bulk density of HAp–CNT composites are given
in Table 1 as a function of CNT content. The condition was
favorable for tissue in-growth in vivo.
Microstructure by FESEM confirmed presence of nanotube
chains, acting as bridges across the HAp grains. The fracto-
graphs revealed that the chains accumulate at the grain
boundaries and help to prevent the intercrystalline propagation
of cracks. This effect is clearly manifested in the enhanced
fracture toughness and flexural strength of the composites over
that of monophasic HAp. The microstructural analysis of the
composites revealed that the non-functionalized CNT chains
were present in agglomerated state around the HAp grains,
while the functionalized chains remain dispersed in the HAp
matrix. FESEM image of one such fractured surface of 1% HAp–
CNT composite sample (Fig. 1) exhibits CNT chains acting as
bridge across a crack moving along the boundaries of HAp
grain, confirming the anticipated hindrance of inter-granular
crack propagation due to presence of ligaments. In pure HAp
matrix, no such bridging action was observed in the fracto-
graph. HRTEM images of composites (Fig. 2) exhibited grains of
HAp adhered to CNTs uniformly. MWCNT hollow chains were
also evident with grains are closely associated with almost no
interfacial gap.
Further, it was also confirmed that all the compositions
were highly biocompatible (as per ASTM F756)(ASTM-F756-13,
2013) and possessed excellent in vitro bioactivity when
immersed in SBF solution for prolonged time period. Different
mechanical properties of the composites have been illu-
strated in Table 2.
Considerable improvement in fracture toughness and flex-
ural strength was obtained for the HAp–CNT composites over
pure HAp. Human cortical bone typically offers a fracture
toughness (K1C) values between 2 and 4 MPa m1/2 depending
on the age of the person (Norman et al., 1995). In the current
study, the 1% HAp–CNT composite achieved a toughness of
1.9 MPa m1/2, which is close to that of human bone. This may be
due to the energy absorbed by the MWCNT chains with the
collapse of concentric layers when subjected to any external
stress, as proposed by Ruoff and Lorents (1995). This energy
absorption mechanism favors the dissipation of energy, thereby
 journal of the mechanical behavior of biomedical materials 60 (2016) 243 –255 247

enhancing the toughness of the composites. Since flexural

strength is the maximum tensile stress that can be sustained
before sample fails, and most ceramic materials fail under
tensile stress before they fail under compressive stress (Green,
1998), the results confirm the load bearing ability of the
nanocomposites for no-articulating bone applications.

3.2. In vivo characterizations

3.2.1. Local inflammatory reactions and healing of wound

In the immediate post operative period, the animals exhib-
ited lameness and mild swelling at the operated site due to
surgical trauma, which gradually subsided and returned to
normalcy within a week. No marked inflammations or other
reactions were observed with either the control or the
composite implants, up to 120 days post-operatively. No
adverse local effects such as marked hematoma or edema
were noticed. The sutures were removed on postoperative
day 10 and the wound healed up gradually.

3.2.2. Radiological analysis

Fig. 1 – FESEM image of fractured surface of HFC1 composites
showing bridging action of CNT chains across the cracks in
HAp crystalline matrix (marked with arrows).
Fig. 2 – HRTEM image of sintered HAp–CNT composite (for
HFC1) with lamellar structure; MWCNT hollow chains were
seen with HAp grains closely associated.
Fig. 3(I–V) exhibited the radiographs of the bony defects with
implants of different groups [HAp (I), HC1(II), HC2 (III),
HFC1(IV) and HFC2 (V)], respectively taken on day 0 (A),
1 month (B), 2 month (C), 3 month (D) and 4 month(E) post-
operatively. The day 0 radiograph with pure HAp implants
(Fig. 3(I)) showed that the cylindrical implant completely
occupied the distal metaphysis of the rabbit femur and
possessed a higher radiodensity than the bone which con-
tinued upto 1 month. The radiograph of 2 month exhibited
smoothening of the implant edges and change of its shape
from cylindrical to oval. The 3 month radiograph showed
reduced density compared to the previous radiographs and
the edge of implant as well as host defect appeared smoother.
Finally, at 4 month, radiograph showed complete bridging of
the cortical defect and a marked reduction in the radiodensity
as compared to that of earlier days. The radiographs with HC1
implants (Fig. 3(II)) showed presence of a well positioned,
highly radiodense implant material at the bony defect on
0 days as well as on 1 month. But on 2 month, a clear
obliteration of the gap between the host and the implant was
observed, associated with reduced radiolucency of the
implant. The radiographs of 3 and 4 month exhibited a
reduction in the size, shape as well as radiodensity of the
implant. As shown in Fig. 3(III), for HC2 implants, the radio-
graph demonstrated well placed radiodense material in distal
femur defect on day 0 that exhibited a gradual reduction of
radiodensity at 1 and 2 month. There was a substantial

Table 2 – In vitro mechanical performance of the different HAp–CNT composites.

Samples Fracture % Change in fracture Flexural % Change in flexural Impact % Change in impact
toughness toughness strength strength strength strength
(MPa m1/2) (MPa) ( J/m2)

HAp 0.81 – 11.23 – 47.25 –

HC1 1.88 132.10 26.28 134.02 133.1 181.69
HC2 1.17 44.44 29.34 161.26 122.95 160.21
HFC1 1.23 51.85 22.58 101.07 78.26 65.63
HFC2 1.7 109.88 21.1 87.89 144.96 206.79
248 journal of the mechanical behavior of biomedical materials 60 (2016) 243 –255

Fig. 3 – Radiographic observation at defect site, taken after 0, 15, 30, 45 and 60 days postoperatively for (I) HAp, (II) HC1, (III)
HC2, (IV) HFC1 and (V) HFC2 implant.

reduction of the radiolucency between bone and implant
along with changes in shape and size of the implant at
3 and 4 month. The bony defects implanted with HAp–CNT
composites with functionalised nanotubes (HFC1 and HFC2),
as shown in Fig. 3(IV) and (V), the day 0 radiographs exhibited
a well placed radiodense graft material that showed a gradual
reduction in the size and shape as well as smoothening of the
edges on 1 and 2 month radiographic-images. The radio-
graphs on 3 and 4 month clearly exhibited a change of the
shape and size of the implant associated with a marked
lowering of the radiodensity of the graft. The cortical con-
tinuity was completely restored.
3.2.3. Microstructural analysis
Figs. 4(A–E) and 5(A–E) exhibited the SEM photomicrographs
of the host implant interface at postoperative 2 months and
4 months, respectively, carried out to study the osseous in
growth process across the interfacial regions. As shown
in Fig. 4A, substantial interfacial gap with little tissue growth
was observed at the host–graft interface with HAp implanted
bone. But in case of the composites, both with functionalised
as well as the non functionalised CNT chains (Fig. 4(B–E)),
there was a considerable reduction in the interfacial gap with
substantial osseous ingrowth indicating superior bone miner-
alization. The new bone regions, marked with yellow circles
 journal of the mechanical behavior of biomedical materials 60 (2016) 243 –255 249

Fig. 4 – SEM micrographs of the host–implant interface at 60 days post-operatively with (A) HAp, (B) HC1, (C) HC2, (D) HFC1 and
(E) HFC2 implants. Both bone, implant surface together with interfacial zones have been demarcated. (For interpretation of the
references to color in this figure, the reader is referred to the web version of this article.)

Fig. 5 – SEM micrographs of the host–implant interface at 120 days post-operatively with (A) HAp, (B) HC1, (C) HC2, (D) HFC1 and
(E) HFC2 implants. Both bone, implant surface together with interfacial zones have been demarcated.

were clearly observed at the interfacial region. For Fig. 4E,
with HFC2 implants, some distinct philopod like structures
were clearly observed coming out from the host bone towards
the graft (shown with higher magnification in the inset)
which represents osteoblast proliferation leading to higher
anchoring of the implant. Fig. 5(A–E) which shows the
micrograph of the host implant interface at 4 months
postoperatively, shows densification of the soft collagenous
tissues leading to strengthening of the interface.
3.2.4. Histological study
Fig. 6 exhibited the histological evaluation of the implanted
bony specimens, at 2 and 4 month postoperatively, carried
out to study the cellular response at the host–implant
250 journal of the mechanical behavior of biomedical materials 60 (2016) 243 –255

Fig. 6 – Histological photomicrograph of implanted bone samples, at 2 and 4 months, showing OB – Osteoblast, OC –
Osteoclast, L – Lacuna, BV – Blood vessel, HC – Haversian canal of HAp; HC1; HC2; HFC1 and HFC2.

Fig. 7 – Fluorochrome labeling images of HAp; HC1; HC2; HFC1; HFC2 at 2 and 4 month post-operatively showing the golden
yellow new bone (NB) and the deep sea green old bone (OB) zones.

interface. As shown in the Fig. 6, the HAp matrix exhibited
solid matrices of bony osteoid with formation of numerous
canaliculi and lacunae. Some portion of periostium was
thickened due to fibroblastic proliferation. The newly formed
blood vessels were well formed to supply the adjacent area of
periosteolar matrix. The spongy portion of bone showed mild
degeneration and formation of bony plate with osteoblastic
and osteoclastic activity. For HC1 and HC2 implants, the
histographs showed well formed osseous tissue with osteo-
blast and osteoclast accumulation in the matrix of osseous
place along with numerous haversian system and bony
spicules. Perichondrium was normal in texture and shape,
containing few osteoblast and osteoclast cells. Angiogenesis
and invasion with osteoclast were quite numerous and some
portion of the bony matrix showed mononuclear cell infiltra-
tion and presence of osteocyte. For HC2, mild fibro-collagen
reaction was observed on the middle portion of bony plates.
HFC1 and HFC2 samples exhibited numerous haversian sys-
tem with lacunae and canalicular spaces. The central cavity
of haversian system was encroached by immature collagen
tissue and the numerous blood vessels penetrated through-
out the matrix. The presence of fair amount of osteoblastic
and osteoclastic activity indicated remodeling of bone. In
comparison to 2 month result, the histology images at
4 months exhibited visibly higher angio-proliferation for all
the implant samples. However, prominent difference in
vascularization was found for HFC1 and HFC2 in comparison
to other samples. It was also been noticed that numerous
haversian system, canalicular structure, infiltration with
fibroblast, osteoblast, and mononuclear cells were present
in all the reinforced samples as compared to pure HAp.
3.2.5. Fluorochrome labeling study
Under UV radiations, the OTC labeled new bone gives char-
acteristic bright golden yellow fluorescence while the old
bone areas gives deep sea green fluorescence. Fig. 7 exhibited
the OTC labelled implanted specimens, collected after sacrifi-
cing the animals at two time point of 2 and 4 months. It was
observed that the bone defects with the composite implants
contain greater extent of golden yellow zones as compared to
 journal of the mechanical behavior of biomedical materials 60 (2016) 243 –255 251

Table 3 – Percentage of bone formation through fluorochrome labeling images at two time point of 2 and 4 months.

Treatment Period of observation

2 Months 4 Months t Significance

HAp 18.109em70.228 22.687dn70.374 9.156 0.01**

HC-1 34.611bm70.312 44.131cn70.529 19.902 0.00**
HC-2 29.939cm70.219 45.170cn70.303 54.132 0.00**
HFC-1 24.790dm70.453 47.619bn70.584 22.052 0.00**
HFC-2 41.853am70.476 52.120an70.277 15.732 0.00**
P-value 0.00** 0.00**

*Pr 0.05.
Means having common superscript in the same column (a, b, c, d and e) did not differ significantly.
Means having common superscript in the same row (m and n) did not differ significantly.
**
Pr 0.01.

the narrower zone in case of HAp implanted defect. The

Table 4 – Push out strength data.
composites promote greater extent of new bone formation
than the phase pure HAp. The images also revealed that the Treatment Period of observation
implants with functionalised CNT chains exhibited maxi-
2 Months 4 Months t Significance
mum golden yellow zone indicating high degree of bone
mineralization and thereby higher osseous activity. It was HAp 15.56cm70.59 18.48cdn70.58  11.13 0.01**
also observed that the extent of new bone formation was HC1 19.55bm70.27 22.45bn70.67  3.14 0.01**
higher for 4 months samples than the 2 months samples, as HC2 14.73cm70.66 17.37dn70.64  12.77 0.01**
evident from the images. Golden yellow fluorescence which HFC1 16.36cm70.72 19.85cn70.56  17.8 0.00**
HFC2 22.57am70.64 24.56an70.67  1.53 0.27
represents bone formation area, measured in mm2 and con-
P value 0.00** 0.00**
verted to percentage of bone formation was statistically
represented in Table 3 at two time point of 2 and 4 months. Means having common superscript in the same column (a, b, c and
The analyses thus seen actually corroborate the observation d) did not differ significantly.
of the images shown in Fig. 7. Means having common superscript in the same row (m and n) did
not differ significantly.
**
P r 0.01.
3.2.6. Biomechanical study
For any biomaterial to be used for direct load bearing
orthopedic applications, the interfacial strength at the 2 months, postoperatively. The plots showed that after
bone–implant interface is an important factor for deciding 4 months postoperatively, HFC2 exhibited the highest inter-
the fate of the implant after the surgery. In any in vivo facial strength both at 2 month (22.57 MPa) and 4 month
experiments, to estimate the biomechanical strength of the (24.56 MPa) postoperatively, as compared to the other com-
samples, their push-out tests were performed at 2nd and 4th positions. HC1 exhibited the second highest value of inter-
month postoperatively. This mechanism is comparable to the facial strength with 19.55 MPa at 2 month and 22.45 MPa at
push and pull out tests of single fibers (Chakraborty et al., 4 month, post operatively. It was also observed that the
2011; Kerans and Parthasarathy, 1991). When a compressive mechanical strength of HC2 and HFC1 was found to be at
load is applied at the top surface of the fiber, the interface par with the control HAp, at 2 and 4 month postoperatively.
undergoes shear stress with the top surface of the fiber
suffering the maximum stress. When the applied load attains 3.2.7. Toxicological study
a critical value, the debonding is initiated. With gradual Fig. 8 represented the histology of hepatocyte and kidney
increase in the applied load, the zone of maximum shear cells, at 4 months postoperatively. For pure HAp implants,
stress gets shifted and the debonding begins to proceed. As histology of the polygonal hepatocyte showed well formed
the load reaches its maxima, the zone of maximum shear central veins, engorged with RBC and mononuclear cells
stress reaches the bottom face of the implant and attains a along with hepatic cords. The hepatic sinusoids contained
critical value. This results in the complete debonding of the RBC, mononuclear cell and very few number of von kupffer
implant and pushes the implant out of the bone matrix. cells, indicating normal structure. For HC1 and HC2 implants,
Push-out strength of the implanted samples was studied the liver cells showed well organized hepatocytes and focal
within 1day after sacrificing the animals, to evaluate the accumulation of mononuclear cell in portal triad. The vascu-
biomechanical strength offered at the bone–biomaterial inter- larity of the whole hepatic parenchyma appeared quite
face. Table 4 represented values obtained for average inter- normal while the sinusoidal spaces contained fair amount
facial strength (calculated as per the method discussed of RBC, mononuclear cells and few von kupffer cells. For HFC1
before) of the implants with statistical analyses. It was found and HFC2 also, the hepatocytes appeared quite normal with
that the interfacial strength of all implants, including the very little infiltration of mononuclear cell around the inter-
control and the composites, was higher at 4 months than at tubular region. The vascular endothelium was engorged with
252 journal of the mechanical behavior of biomedical materials 60 (2016) 243 –255

Fig. 8 – Histological section of rabbit liver at 4 month postoperatively, showing CV – Central Vein, H – Hepatocyte Cells, V – Von
Kupffer cells, S – Sinusoids of A – HAp; B – HC1; C – HC2; D – HFC1; E – HFC2 and photomicrograph of kidney, at 4 month
postoperatively, showing B – Bowman's Capsule, CD – Collecting Duct, PD – Proximal Duct, of A – HAp; B-HC1; C- HC2; D – HFC1;
E – HFC2.

R.B.C and few mononuclear cells. Distinct Von Kupffer cells in
sinusoidal spaces were also quite few in numbers.
In HAp, the medullary portion of kidney was found to be
well prominent with their tubules and the renal matrix showed
well vascularity all throughout the section. The ascending
Loops of Henle appeared normal in size and shape while the
collecting ducts appeared dilated with colorless fluid. The renal
parenchyma of HC1 and HC2 showed normal tubular architec-
tural pattern with well set up columnar epithelium all around
the tubules. Intertubular spaces showed accumulation of RBC
and formation of new endothelial cells indicating quite active
phase of renal function. Numerous angiogenesis were observed
around the intertubular spaces. Epithelium of the lining tubules
was normally placed and contained very few numbers of R.B.C
and mononuclear cells. Kidney of HFC1 and HFC2 showed
symmetrical arrangement of renal tubule with high vascular
pattern indicating active stage of absorption and reabsorption
procedures. The collecting tubules also contained epithelium
cell in synchronized pattern. The interlobular region was
infiltrated by few mononuclear macrophages and RBC. Angio-
genesis was very less.
4. Discussions
Concentric graphene layers of MWCNT are held together by
van der Waals attractions and are arranged at an interlayer
distance of 0.3–0.4 nm. In their native state, these inherent
attractions between the nanotube walls keep them in aggre-
gated bundles, resulting in poor solubility and processability
and act as main obstacle for different applications further
(Eitan et al., 2003; Hirsch, 2002). Side chain functionalisation
on the MWCNT surface improves solubility and enhance their
 journal of the mechanical behavior of biomedical materials 60 (2016) 243 –255
dispersability (Eitan et al., 2003). A variety of functional
groups or molecules introduced through chemical modifica-
tion are reported (Lei et al., 2011; Sun et al., 2002). But, in this
case, we have reported through XRD and FTIR that carbox-
ylation of MWCNT surface occurs through this acid treatment
process. In a nutshell, pristine chains exhibited single peak at
2359 cm  1, corresponding to the C¼C stretching while the
treated CNT chains give characteristic peaks at 1053, 1226,
1718, 2854, 2926 and 3375 and 3722 cm  1. The broad band
around 3375 and 3722 cm  1 is attributed to the presence of
water and hydroxyl functional groups (νOH) that may have
resulted from the carboxylation process.
For any material to be used as a bone graft, not only it should
provide sufficient physical and mechanical integrity to be able to
function under loaded conditions, it should also conform to the
biocompatibility issue. In vivo animal study is the only method of
analyzing the material's tissue response. In general, CNTs have
excellent mechanical properties and strength, but having toxicity
due to its dissolution property. In other side, HAp has good
biocompatibility, it lacks the strength. These advantageous and
disadvantageous properties of both materials can be comple-
mented to each other by making a composite that would
improve the required properties (Rajesh et al., 2012).
In this study, all the implants were well placed at the defect
site and well accepted, without causing any serious inflamma-
tion in surrounding tissues. Healing was uneventful and there
was no evidence of adverse local effect like hematoma or
edema indicating acceptance of the implant. In HAp implanted
bone, sequential radiographic analysis showed gradual redu-
cing radiodensity of implant and marked reduction in the
radiodensity at 4 month, indicating that though the implant
was not absorbed but undergone structural changes due to host
graft interaction. In HC1 and HC2 implant, the radiodensity of
graft material was more close to that of host bone, indicating
formation of well organized bony callus and thereby good
progress of healing process, without any noticeable change in
implant material. The cortical continuity was completely
restored in with HAp–CNT composites with functionalised
nanotubes (HFC1 and HFC2) indicating better response.
Study of new bone formation can be confirmed through
different labeling techniques that uses specific bone markers
(van Gaalen et al., 2010). In the fluorochrome labeling method
used in our work, oxytetracycline (OTC) with fluorescence
property in UV light, is used as the bone forming marker that
helps to quantify the newly formed bone (Kovar et al., 2011).
OTC follows the path of the ionized calcium [Caþ2] and gets
deposited in areas of active mineralization (Gibson et al.,
1978). The labeled new bone and old bone discharge bright
golden-yellow and dark, sea green fluorescence respectively
when observed under UV light (Dahners and Bos, 2002). HC1
and HC2 samples showed moderate bone formation only in
central zone. The HAp–CNT (HFC1 and HFC2) composites
samples exhibited increased amount of primary bone in-
growth in both central and peripheral zone at two time
points as evidenced by more golden yellow fluorescence than
phase pure HAp. This increased amount of new bone deposi-
tion may be attributed to the increase in mineral apposition
rate, which may be due to presence of the reinforcing phase
in monophasic HAp matrix.
253
Both histomorphometry and microstructures (by SEM)
exhibited high osseointegration capability of composites as
compared to control. Histograms exhibited well developed
Haversian canal and osseous canaliculi along with osteoblas-
tic proliferation and angiogenesis. SEM micrographs revealed
enhanced osseous integration at host–graft interface of com-
posites as compared to control. Osteoid formation along the
transplanted material is clearly visible, confirming prolifera-
tion of more osteoblastic cells and subsequent bone matrix
deposition (Perrien et al., 2002). Composites with functiona-
lised nanotubes exhibited growth of soft collagenous tissue
from host surface towards the implant surface at 2 month
postoperatively, indicating excellent host bone interaction.
CNTs could decrease osteoclast number to inhibit bone
resorption (Abarrategi et al., 2008; Tonelli et al., 2012). More-
over, CNTs did not have cytotoxicity to osteoblasts and did
not have harmful effects on osteoblast differentiation or
mineralization (Li et al., 2008; Verdejo et al., 2009). The
interfacial gap was reduced and soft collagens develop into
dense bony matters in 4 months postoperatively. Intercon-
nected porosity is observed for composite implants that are
supposed to allow osteoprogenitor cell penetration, cell
migration, and cell-attachment, thereby enabling new bone
in growth and strengthening the bone implant interface
(Damien et al., 2003). These interconnected porosities also
provide higher surface area for enhanced mechanical inter-
locking between implant and host tissue and path for micro-
nutrients (Ramay and Zhang, 2004; Yang et al., 2001).
Push out tests revealed that the biocomposite implants
were very stable as far as long term application is concerned
with considerably high interfacial strength suitable for any
load bearing applications (Wang et al., 2014). The higher
mechanical strength of the composite implant can be
endorsed to the increased CNTs at the joining microsphere–
microsphere areas (Mikael and Nukavarapu, 2011). In this
study, the high value of interfacial strength of HFC2 and HC1
may be attributed to the excellent tissue in-growth at the
host bone–implant interface, as confirmed from the SEM and
radiological evidences. In another study, it has also been
observed that addition of CNT on HAp not only increased the
bio-mechanical properties effectively but also promoted cell
growth on osteoblast cell (Lahiri et al., 2010; Xu et al., 2009).
Since carbon nanotubes, belonging to the carbon family, are
a well known toxic material and are known to affect the
respiratory system when used in large proportions (Mercer
et al., 2010). For successful application of CNTs reinforced
biomaterial in the biomedical field, it is of imperative necessity
to assess the bio-distribution of CNTs and to evaluate their
influence on organs or tissues (Mercer et al., 2010). In this study,
toxicological effects of the HAp–CNT biocomposites were per-
formed on two vital excretory organs of the body – the kidney
and the liver cells. Histology of neither the kidney nor the liver
cells exhibit any abnormal structure, thereby confirming that
presence of carbon nanotubes (upto 2 wt%) as a reinforcing
phase in the hydroxyapatite matrix do not evoke any toxicity in
the body (Arsecularatne and Zhang, 2007).
254 journal of the mechanical behavior of biomedical materials 60 (2016) 243 –255
5. Conclusions
The present study examines the in vivo evaluation of HAp–
CNT composites implants on physic-mechanical, and in vivo
osteogenesis properties. All the HAp–CNT composites, with
both non-functionalised as well as functionalised CNT
chains, showed considerable in vivo new bone formation
during the four months period of study. Based on the
microstructural, histological, radiological and fluorochrome
labeling results, CNT doped HAp composites showed
enhanced early-stage bone formation at the rabbit model
defect site. One of the non functionalised CNT composites
and one functionalised CNT composite with HAp proved to be
more effective than the others as well as the control. Our
study suggests that incorporation of CNT as a reinforcing
material in the HAp matrix effectively enhances early stage
in vivo osseointegration and bone remodeling properties
along with enhancement of the mechanical properties of
the monophasic HAp ceramics.
Disclosure
The authors declare that there is no conflict of interest with
any financial organization regarding the material discussed
in the manuscript.
Acknowledgment
The authors would like to thank University Grants Commis-
sion and Council of Scientific and Industrial Research for
financial support (Vide no. 09/096(0617) 2K10-EMR-1). Thanks
are also due to the Director, CSIR-Central Glass and Ceramic
Research Institute, Kolkata, India and Vice Chancellor, West
Bengal University of Animal and Fishery Sciences, Kolkata,
India for their generous and kind support to this work. All the
personnel involved in the characterization of the materials
are sincerely acknowledged.
Appendix A. Supplementary material
Supplementary data associated with this article can be found
in the online version at http://dx.doi.org/10.1016/j.jmbbm.
2016.02.005.
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