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Layered double hydroxide induced advancement in

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joint prosthesis using bone cement: the effect of

Cite this: J. Mater. Chem. B, 2013, 1,
2275 metal substitution†
Govinda Kapusetti,a Raghvendra Raman Mishra,b Swati Srivastava,c Nira Misra,a
Vakil Singh,d Partha Roy,c Santosh Kumar Singh,e Chanchal Chakraborty,f
Sudip Malikf and Pralay Maiti*g

Received 2nd January 2013
Accepted 28th February 2013
DOI: 10.1039/c3tb00004d
www.rsc.org/MaterialsB
Poly(methyl methacrylate) based bone cement and its nanocomposites with layered double hydroxide
(LDH) have been developed with greater mechanical strength and biocompatibility as a grouting
material for total joint arthroplasty. Bivalent magnesium has been replaced with trivalent aluminium
with various mole ratios, keeping the layered pattern of the LDH intact, to cater for the effect of varying
substitution on the property enhancement of the nanocomposites. The intercalation of polymer inside
the LDH layers makes them disordered and mechanically stiffer and tougher by more than 100%. The
thermal stability of bone cement has increased by more than 30
C in the presence of 1 wt% of
nanoLDH, homogenously distributed in the bone cement matrix by creating an inorganic thermal
barrier out of the LDH dispersion. The improvement in the properties of the nanocomposites has been
explained in terms of the strong interaction between nanoLDH and polymer. The superior bioactivity
and biocompatibility of the nanocomposites, as compared to pure bone cement, has been established
through hemolysis assay, cell adhesion, MTT assay and cell proliferation using fluorescence imaging. The
developed nanocomposites have been used as a grouting material and significant improvements have
been achieved in fatigue behaviour with gradual increment of Al substitution in the Mg : Al mole ratio
in nanoLDH, demonstrating the real use of the material in the biomedical area. In vivo experiments on
rabbits clearly revealed the superior efficacy of bone cement nanocomposites, over pure bone cement
and a blank.

Introduction bone–implant interface from everyday activities and the

strength of the bone cement is somehow proportional to
Poly(methyl methacrylate) (PMMA) based bone cement has the joint's life/durability.2 All commercial PMMA based bone
been introduced as a grouting material for total joint replace- cements are cold cured powder–liquid combined systems, and
ment surgeries.1 The space between the bone and implant can for clinical applications, powder and liquid components are
be lled with bone cement for stronger mechanical bonding to combined and the mixture is inserted into bone where the
prevent destabilization during stress distribution between the polymerization reaction takes place. PMMA bone cement has
the ability to resist fatigue related cracking to a certain extent.3
a
School of Biomedical Engineering, Indian Institute of Technology, Banaras Hindu The typical success rate of joint surgery is usually around 10–
University, Varanasi 221 005, India 20 years. More than 75% of failures are due to aseptic loosening
b
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, which is attributed to impact induced failures in the cement
Varanasi 221 005, India
c
mantle during cyclic loadings (fatigue) arising from poor
Department of Biotechnology, Indian Institute of Technology Roorkee, 247667,
Roorkee 247667, India
mechanical properties of bone cement4,5 and lack of bone
d
Department of Metallurgical Engineering, Indian Institute of Technology, Banaras adhesiveness (non-bioactivity).6,7 Attempts are being made for
Hindu University, Varanasi 221 005, India new formulations of bone cement to improve fatigue strength to
e
Centre of Experimental Medicine and Surgery, Institute of Medical Sciences, Banaras achieve better lifespans of the joint surgeries. Fracture tough-
Hindu University, Varanasi 221 005, India ness usually increases with increasing molecular weight of the
f
Polymer Science Unit, Indian Association for the Cultivation of Science, Jadavpur, polymers.8,9 However, increase in viscosity makes it difficult to
Kolkata 700 032, India
g
insert bone cement in to cancellous bone during implantation.
School of Materials Science and Technology, Indian Institute of Technology, Banaras
Hindu University, Varanasi 221 005, India. E-mail: pmaiti.mst@itbhu.ac.in
In addition, it may inhibit the mechanical interlocking between
† Electronic supplementary information (ESI) available. See DOI: bone and the implant. Reinforcement of bone cement by bres
10.1039/c3tb00004d or particles might be an alternative technique to improve the

This journal is ª The Royal Society of Chemistry 2013 J. Mater. Chem. B, 2013, 1, 2275–2288 | 2275

Journal of Materials Chemistry B
fracture toughness of bone cement. Various new formulations
of bone cements with different ller materials, like g-meth-
acryloxypropyltrimethoxysilane and calcium acetate,10 b-TCP
particles encapsulated with poly(ethylene glycol),11 oligomer,12
polyethylene bers,13 carbon bers,14,15 hydroxyapatite,16 tita-
nium bers,17,18 glass19 and graphite bers20 have been devel-
oped. In our previous work, the solvent derived bone cement
has been incorporated with organically modied clay21 and an
improvement of 30% in toughness was achieved at a lower clay
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concentration.
The concept of the reinforcement of nanollers into the
polymer matrix as an effective technology for obtaining
improved and unique properties of the hybrids, using a
minimum amount of llers, has already been well documented.
In polymer composites, nanometer dimension layered crystal-
line materials as llers, promote the intercalation and/or exfo-
liation by polymer chains and this phenomenon results in
enhancement of mechanical and other properties. In recent
years, researchers have been attracted towards synthetic clays,
mainly layered double hydroxides (LDHs), because of their
structural similarity with natural clay minerals, such as mont-
morillonite and their versatile properties. Layered double
hydroxides (LDHs) have been identied for their applications in
biomedicine,22,23 pharmaceutics24 and heat stabilizers due to
their favourable biological properties, such as low toxicity, good
biocompatibility and ease of manufacturing. The general
formula of a LDH is M1xIIMxIII(OH)2(An)x/n$mH2O, where x
varies from 0.20 to 0.33;25 MII is a divalent metal ion like Mg+2,
Zn+2; MIII is a trivalent metal ion like Al+3, Cr+3 and A is a
counter anion with valency n, like CO32 or Cl to balance the
charge resulting from the metal substitution. MgAl–CO3 is one
of the important classes of LDH. Bivalent Mg plays an important
role in changes in bone matrix that determine bone fragility.26
Magnesium implanted aluminium, through the ion implanta-
tion technique, shows considerable improvement in the
attachment and spreading of human cells.27 Hence, Al
substituted LDH can be a potential material as ller for bone
cements to be used for bone implant/surgery. So far, there is no
systematic report of the gradual substitution of bivalent cations
with trivalent metal ions and their composites related to the
effect of metallic substitution on the properties of the
composites.
In the present work, a series of LDH with varying Al substi-
tution have been synthesized and dispersed in bone cement
matrix through in situ polymerization to examine the changes in
properties at varying Mg : Al mole ratios. The changes in the
mechanical and thermal properties of the nanocomposites have
been studied and the results are discussed in terms of interac-
tion which in turn changes with Al substitution. The biocom-
patibility of LDH nanocomposites has been studied in detail
with hemolysis, cell adhesion, cell viability, MTT assay and
uorescence imaging. Finally, the developed nanocomposites
have been applied as a grouting material in real bone implants
and their fatigue behaviour has been examined. Signicant
improvement was observed in the resistance against fatigue
failure with increasing Al substitution in LDH. Thus, the best
possible mole ratio of Mg : Al in LDH has been established to
2276 | J. Mater. Chem. B, 2013, 1, 2275–2288
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prepare nanocomposites of bone cement based on the
combined results of thermal behaviour, mechanical properties
and biocompatibility. In vivo testing has been performed to
exhibit the strength of newly developed biocompatible
nanobiohybrids.
Experimental
Materials
Commercially available bone cement Depuy CMW-1 (Jhonson &
Jhonson, UK) was used in this work. Bone cement is a two pack
system containing nely divided white powder and colourless
liquid. Magnesium nitrate hexahydrate of 99% purity,
aluminium nitrate nonahydrate (98%), sodium carbonate (99%)
and sodium hydroxide were obtained from Merck India Ltd.
Synthesis of Mg–Al layered double hydroxides
The co-precipitation method was used for preparing different
molar ratios of carbonate intercalated Mg–Al layered double
hydroxides (LDH). Salt solutions with Mg : Al molar ratios of
2 : 1, 3 : 1, 4 : 1, 5 : 1 and 10 : 1 were prepared by adding
Mg(NO3)2$6H2O of 2.2, 3.3, 4.4, 5.5 and 10.1 mmol, respec-
tively, to Al(NO3)3$9H2O of 1.1 mmol in 75 ml deionised water.
250 ml Na2CO3 solution of 2.3 mmol was prepared in de-
ionized water and was kept in a ve neck at bottom ask. The
ask was integrated with two burettes, one condenser, one gas
inlet–outlet and one pH meter probe. The solution was de-
gassed by purging puried nitrogen. The whole solution was
stirred vigorously and previously prepared salt solutions of
Mg(NO3)2$6H2O of 2.2, 3.3, 4.4, 5.5 and 10.10 mmol and
Al(NO3)3$9H2O of 1.1 mmol to prepare molar ratios of Mg : Al
of 2 : 1, 3 : 1, 4 : 1, 5 : 1 and 10 : 1 in 75 ml deionised water
were added drop wise in the Na2CO3 solution. One molar
NaOH solution was added drop wise simultaneously to the
above solution to maintain constant pH  11. LDH was well
crystallized in the range of pH  8 to 10.5. However, at a
higher pH (11), LDH was still precipitated, but the particle
size was noticeably reduced.28 The whole solution was stirred
at 65
C for 12 h followed by separation of a white coloured
precipitate from the solution by centrifugation (2500 rpm),
washed several times with de-ionized water and dried in an
oven at 45
C for 36 h.
Preparation of bone cement nanocomposites
Pure bone cement was prepared by mixing the powder and
liquid components manually with the help of a glass rod in a
Petri dish for 2 minutes at room temperature. Nanocomposites
were prepared using 1 wt% of LDH having different Mg : Al
molar ratios. The powder part of the bone cement and LDH
were mixed thoroughly in an electronic mixer at 2000 rpm for
15 min and was added to the liquid component of the bone
cement. The nal mixture was stirred vigorously with the help of
a glass rod for 2 minutes at ambient temperature. Henceforth,
the notation of BC-0, BC-x : y (x ¼ Mg and y ¼ Al) will be used for
the pure bone cement and 1 wt% nanocomposites with
different LDHs, respectively. The ratio aer BC indicates the
This journal is ª The Royal Society of Chemistry 2013

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molar ratio of Mg : Al in nanoLDH used for preparation of
nanocomposites. Thus, BC-2 : 1, BC-3 : 1, BC-4 : 1, BC-5 : 1 and
BC-10 : 1 represent nanocomposites of bone cement with 2 : 1,
3 : 1, 4 : 1, 5 : 1 and 10 : 1 Mg : Al mole ratios in LDH,
respectively.
Characterization
X-ray diffraction
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The degree of intercalation and/or exfoliation of LDH in the
presence of bone cement was examined by using an advance
wide-angle X-ray diffractometer with Cu Ka radiation and a
graphite monochromator (wavelength, l ¼ 0.154 nm, Bruker
AXS D8, Germany). Thin sheets of the samples were placed on a
quartz sample holder at room temperature and were scanned at
diffraction angle 2q from 1
to 50
at scan rate of 1
min1.
TEM
The nanoparticle dispersion in the matrix was checked by using
TEM (JEOL, 2010 EX) operated at an accelerating voltage of 200
kV. A thin layer, around 70 nm thick, from the nanohybrid
sample was sectioned at 70.0
C using a Leica ultra-microtome
equipped with a diamond knife.
UV-Visible spectroscopy
The UV-Visible measurements were carried out by using Shi-
madzu (UV-1700), Pharma Speck, UV-Vis spectrophotometer
operating in the spectral range of 200–1100 nm. Transparent
thin lms of 30 mm thickness were prepared using bone
cement and its nanocomposites by dissolving in THF solvent in
a Petri dish.
FTIR
FTIR technique was applied to detect the functional groups and
to understand the nature of the interactions between the bone
cement and LDH. FTIR was performed in the absorbance mode
at room temperature from 400–4000 cm1 using Nicolet 670
FTIR with a resolution of 4 cm1. The bubble free thin lms
were prepared by dissolving in THF solvent with special care.
TGA
The thermal stability of the bone cement and its nano-
composites was examined by using a thermogravimetric
analyzer (TGA, Mettler-Toledo) tted with a differential thermal
analyzer (DTA). Data was taken from 40–550
C. All the experi-
ments were performed at a heating rate of 20
C min1 in
nitrogen atmosphere.
DSC
The thermal behaviour of the pure bone cement and its nano-
composites was determined via DSC using Mettler 832 at a
scan rate of 10
C min1. The DSC was calibrated with indium
before use.
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Journal of Materials Chemistry B
Mechanical properties
The Young's modulus and toughness were determined using
dog bone shaped samples, prepared by the compression
moulding technique, using an Instron 3369 tensile testing
machine. Samples were stretched uniaxially at a rate of 5 mm
min1 at room temperature. Several samples were tested for
error estimation.
Fatigue testing
High cycle fatigue testing was performed on the bone cement
and its 1 wt% LDH nanocomposites. The designed experi-
mental setup mimics an in vivo human hip joint. Human male
femurs of almost comparable mechanical properties were
collected from the Institute of Medical Science, Banaras Hindu
University, Varanasi, and the metallic stems were purchased
from ORMAD medical technologies Ltd, India. The specimen of
the implanted hip is shown in the ESI Fig. S1.† Two implanted
femurs were tested under axial fatigue using a servo hydraulic
MTS of 50 kN capacity (model 810) interfaced with test star IIS
control. The applied loads on the implanted femur bone started
from a load equal to the human gait cycle to failure of the joint.
The load was applied at the angle of 45
with the help of a
special design set up to mimic the human hip joint (ilium), as
shown in the scheme. At every load level, 50 000 cycles were
applied at 3 Hz frequency and loads of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,
4.0 and 4.5 kN were applied step by step. (ESI Fig. S1† shows the
whole experimental set up of the system).
Contact angle
Determination of the contact angle for implant material is
essential to examine their hydrophilicity. Contact angles of
bone cement and its various nanocomposites were measured
with a Kruss F-100 tensiometer. Three specimens of each
sample in the form of thin strips (1  10  20 mm3) were
subjected to contact angle measurement in water. The accuracy
of the contact angle measurement is 1
.
Hemolysis assay
A hemolysis assay was carried out on the heparinized lms29 of
bone cement and 1 wt% of LDH nanocomposites with acid
citrate dextrose (ACD) human blood in triplicate. 5 ml of ACD
blood was prepared by adding 4.5 ml of fresh human blood to
0.5 ml ACD. The ACD solution was prepared by mixing 0.544 g
anhydrous citric acid, 1.65 g of trisodium citrate dihydrate and
1.84 g of dextrose monohydrate to 75 ml of distilled water.
Heparinized lms of bone cement and its various nano-
composites with equal weights were taken in different test tubes
followed by the addition of 10 ml of phosphate buffer solution
(PBS). Two different test tubes with 10 ml distilled water and 10
ml PBS solutions were taken as positive and negative controls,
respectively. All these test tubes were kept in desiccators for 30
min at 37
C, followed by adding 0.2 ml ACD blood to all the test
tubes and incubating for 1 h at 37
C. Aer incubation, the test
tubes were centrifuged at 5000 rpm for 8 min. Optical density
(OD) was measured at 545 nm from 1 ml supernatant with
J. Mater. Chem. B, 2013, 1, 2275–2288 | 2277

Journal of Materials Chemistry B
Shimadzu (UV-1700), Pharma Speck, UV-Vis spectrophotom-
eter. The percent of hemolysis was calculated as follows:
OD of the test sample  OD of the negative control
% of hemolysis ¼
OD of the positive control  OD of the negative control
Specimen for cell culture studies
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Bone cement and its 1 wt% LDH nanocomposites were selected
for in vitro cell culture studies with specimen sizes of 10  10  4
mm3. A Teon sheet of 4 mm thickness was moulded with a
male–female puncture system and the moulds were smoothened
with emery paper. Samples were injected into the square shaped
Teon sheet cavities and the injected Teon mould was clamped
with a vice for 10 min and covered with two plain Teon sheets
above and beneath. Cured specimens were removed from the
moulds and the edges were smoothened with emery paper and
the specimens were stored at room temperature. Before per-
forming the cell studies, the specimens were washed with iso-
propanol for removing the attached debris. For surface
sterilization of the specimens, each specimen was washed thrice
with phosphate buffered saline (pH  7.2) and was exposed
under UV light for 8 h. Cement extracts (cement leached medium)
were then prepared for the microscopic analysis of cellular
structures. As per USTM standards, the weight/volume ratio of
cement and medium was kept at 1 g/5 ml for an incubation
period of 72 h. The cement specimens or its nanocomposites
were incubated in minimum essential medium (MEM) at 37
C
for 72 h in glass containers. On completion of the incubation
period the extracts were collected and stored at 20
C.
Cell culture
MG-63 is an established cell line of osteoblast-like cells. It was
obtained from the National Centre for Cell Sciences, Pune, India
(Ref. no. CREP696). Cells were cultured in MEM supplemented
with heat-inactivated fetal calf serum (10%) (Invitrogen Carls-
bad, CA, USA), penicillin (100 U ml1), streptomycin (100 U ml1)
and gentamicin (20 mg ml1) (from Sigma, St. Louis, MO, USA) at
37
C in a humidied CO2 incubator maintained at 5% CO2.
Cell adhesion
The cell adhesion behaviour of the MG-63 cell line to the bone
cement and its 1 wt% nanocomposites was evaluated through a
modied crystal violet staining assay protocol.30 For this assay,
1  105 cm2 cells were seeded on the cement specimen surface
and incubated in a CO2 incubator for 4 h to examine the cell
adhesion properties of the matrix. Aer 4 h of incubation, the
specimens were washed with PBS to remove the unattached
cells and then the attached cells were xed with ice-cold 4%
paraformaldehyde for 20 min. Cell permeabilization was carried
out with 20% methanol for 20 min aer PBS washing. The
attached cells were then stained using 0.5% crystal violet
aqueous solution (Raymond Lamb, UK) for 30 min. Excess
stains were removed by three gentle washes in deionised water
followed by elution of the residual crystal violet with 10% acetic
2278 | J. Mater. Chem. B, 2013, 1, 2275–2288
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acid for 30 min during gentle agitation on an elliptical shaker.
Optical density (OD) of the eluted solution was measured in a
 100
UV-Vis spectrophotometer (double beam LI-2800, Lasany, India)
at a wavelength of 570 nm, with the background absorbance
value measured at 650 nm. The optical density values obtained
correlate directly with the attached cell number.
Cell viability
Cell viability was measured by MTT (4, 5-dimethylthiazol-2-yl-
2,5-diphenyl tetrazolium bromide) assay.31 For this assay, 0.5 
105 cm2 MG-63 cells were seeded on the cement specimens in
24 well culture plates with 1 ml MEM and incubated for 24, 48
and 72 h time intervals at 37
C. Following this treatment, 50 ml
of 5 mg ml1 MTT solution was added to the wells and incu-
bated for another 4 h at 37
C to form formazon. The MTT-
containing medium was then aspirated and 100 ml of DMSO
(Himedia, Mumbai, India) was added to it to solubilize water-
insoluble formazon. The absorbance was taken using a UV-Vis
spectrophotometer. The % cell viability was calculated using the
formula:
OD of T
% of cell viability ¼  100
OD of C
where, C ¼ optical density of ‘control’ representing osteoblasts
incubated in medium alone and T ¼ optical density of test
specimen representing osteoblasts treated with the corre-
sponding extracts.
Fluorescence microscopy
The extracts of each type of cement/nanocomposites and a
control group were visually examined to characterize cell
proliferation using uorescence microscopy aer 24 h of incu-
bation. The MG-63 cells were suspended in fresh complete
medium, at a density of 0.5  105 cells per ml in a 96 well plate
of triplicate and were incubated for 4 h. Aer initial incubation,
0.2 ml extracted medium was replaced with fresh medium in
each well, except the control, and was nally incubated for 24 h.
Cells were washed with PBS, stained with propidium iodide and
incubated in a dark place for 30 min at room temperature.
Imaging was performed using a Nikon Eclipse 80i uorescence
microscope (Nikon, UK).
In vivo studies
A total of 18 male rabbits weighing approximately 2.5–3.0 kg
were procured from the animal care unit, Centre of Experi-
mental Medicine and Surgery, Institute of Medical Sciences,
Banaras Hindu University, Varanasi aer taking prior permis-
sion of the institutional ethical committee vied reference no.
Dean/12-13/CAEC/25. Three groups (blank, BC-0 and BC-2 : 1) of
six rabbits were taken for each material intended for
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Scheme 1 During surgery (a) drilling holes on the rabbit tibia and (b) filling with
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bone cement/composites.
implantation. During the surgical performances each rabbit
was anesthetized using intra peritoneal injections of ketamine
hydrochloride (1 mg kg1), 1 mg kg1 of midazolam supple-
mented with 2% xylocaine with adrenaline 1 : 1000 under the
standard conditions of animal care guidelines. The right femur/
tibia of the fully anesthetized rabbit was washed with iodine
tincture (Betadine 10% aqueous) followed by alcohol (70%).
Two holes were created by using a specially designed hand
drilling apparatus with 4 mm diameter and 10 mm depth.32
Rabbits of group 2 and 3 were injected with pure bone cement
(BC-0) and nanocomposite (BC-2 : 1), respectively. Group 1 was
taken as a control (without any material). The incise area was
covered with a micro pore with 3–5 stitches. The scheme 1
shows the drilled holes and the image aer lling the holes with
the cement/nanocomposite of the tibia of rabbit. The X-ray
images have been taken every 5 days from the day of post
surgery. Right hind limbs of each group rabbit were processed
for digital X-ray radiography (SHIMADZU RAD Speed, at 41 KV)
Fig. 1 (a) XRD patterns of LDHs with varying indicated Mg : Al ratios, (b) FTIR spectra of MgAl–CO3 with different molar ratios of Mg : Al as indicated, and (c) XRD
patterns at a higher angle of indicated LDHs.
This journal is ª The Royal Society of Chemistry 2013
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Journal of Materials Chemistry B
to examine the osteo changes as a function of time. Radio-
graphs were focused on three regions of interest; proximal
metaphyseal, diaphyseal region and distal metaphyseal.
Statistical analysis
The results are expressed as mean values (SE). The analysis of
variance followed by a post hoc Dunnett's testing was per-
formed for the hemolysis assay and cell adhesion and Bonfer-
roni's method was performed for the cell viability tests.
Dunnett's method was used for single comparisons while
Bonferroni's method was used for multiple comparisons in
ANOVA. In all cases, P < 0.05 was considered to be signicant.
GRAPH PAD PRISM version 5.1 was used to perform statistical
analysis. All the tests and observations were made in triplicate
and means of those values are reported here. The data was
checked for normal distribution and equality of variance and
was log transformed wherever it was found to be necessary.
Results and discussions
LDH with varying metallic substitution
The gradual replacement of divalent Mg with trivalent Al creates
a higher surface charge density in the interlayer in order to
balance the charge. The nanostructure and structure of LDH
with varying Mg : Al ratios were determined through X-ray
diffraction patterns. Fig. 1a shows diffraction peaks at 2q of
5.96
, 5.85
, 5.78
and 5.58
for 2 : 1, 3 : 1, 4 : 1 and 5 : 1 Mg : Al
LDHs, respectively, for (001) planes and their corresponding
J. Mater. Chem. B, 2013, 1, 2275–2288 | 2279

Journal of Materials Chemistry B
d-spacings are found to be 1.48, 1.51, 1.53 and 1.59 nm, indi-
cating larger d-spacing with an increasing Mg/Al molar ratio
presumably due to greater inter-layered water molecules with a
lower substitution of Mg with Al. The absence of any peak in
10 : 1 LDH is due to the disordered layered structure with the
lesser substitution of Mg with Al. It is necessary to mention here
that pure Mg(OH)2 exhibits a brucite layered structure (hexag-
onal) but it does not show any peak at a lower angle (<10
)
assigned to the (001) plane.33 Lower substitution of Mg with Al
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or a higher Mg : Al mole ratio (10 : 1) also exhibits similar
behaviour, while greater substitution or a lower Mg : Al ratio
reveals a stacked pattern and well dened peak in XRD,
presumably due to the ordered nature of LDH at higher
substitution. Fig. 1b shows the FTIR spectra of different molar
ratios of carbonate anion intercalated MgAl–LDH. An intense
FTIR band at 3471 cm1, attributed to the O–H stretching
vibration of interlayer water molecules, with an increasing
Mg : Al ratio in LDHs further supports the greater intercalation
of water molecules for a higher Mg : Al mole ratio (Fig. 1b).
Moreover, the shoulder band around 3030 cm1 for a lower
Mg : Al ratio or greater substitution of Mg with Al, due to
hydrogen bonds between the interlayer water molecules and
interlayer CO32 anions,34 gradually disappear for higher
Mg : Al ratios or lower substitution of Mg with Al indicating a
lesser extent of hydrogen bonding. So, the interlayer water
molecules are relatively free in higher Mg : Al LDH causing
slightly greater d-spacing while tightly bound water molecules
in lower Mg : Al LDHs through hydrogen bonding makes the
gallery spacing lower. Even though the greater substitution of
metal (Mg) with Al (e.g. Mg : Al  2 : 1) creates more CO32
intercalated anions to counter balance the charge in the inter-
layer the gallery spacing decreases primarily due to stronger
hydrogen bonded water molecules in the interlayer. Further, the
sharp and intense vibration band at 1372 cm1, assigned to the
n3 asymmetric stretching mode of interlayer CO32 anions,
gradually shied to a higher wavenumber range with increasing
Mg : Al ratio indicating greater interaction presumably with
inter layered water molecules. The peak positions are 1372,
1373, 1375, 1382 and 1388 cm1 for 2 : 1, 3 : 1, 4 : 1, 5 : 1 and
10 : 1, respectively. The band at around 1640 nm is due to the
bending vibration of the interlayer water. Some small peaks
appeared between 800 and 400 cm1, due to the lattice vibration
of M–O and O–M–O (M ¼ Mg, Al) groups.35 Thus, FTIR studies
conrm formation of MgAl–CO32 LDH and the interaction
with interlayer water molecules as a function of Mg : Al ratio or
varying substitution of Mg with Al which dictate the gallery
spacing as observed in XRD patterns.
Fig. 1c shows the higher angle XRD patterns of varying
Mg : Al LDHs. All the samples exhibit the characteristic
diffraction peaks of MgAl–CO32–LDH.34 From the peak index-
ing, the ascribed structure is predicted as a hexagonal unit cell
with rhombohedral symmetry36 and the whole pattern was
matched with the JCPDS le. The diffraction peaks of the 2 : 1
molar ratio LDH appears at 12.3
, 23.8
, 35.2
, 39.2
, 46.7
,
60.5
, 62.1
and 66
corresponding to the (003), (006), (009),
(015), (018), (110), (113) and (116) planes and similar peaks have
been observed for other samples as well, conrming the
2280 | J. Mater. Chem. B, 2013, 1, 2275–2288
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formation of a crystalline layered structure and the in-plane
diffraction peaks for (110), (113) and (116) crystal planes reveal a
good dispersion of metal ions in the hydroxide layers.37 The
diffraction peaks were shied slightly towards lower angles with
an increasing Mg : Al molar ratio indicating a higher interlayer
distance for the lower substitution of Mg with Al, similar
phenomena were observed in the nanostructures in Fig. 1a.
Nanostructure
Fig. 2a shows the XRD patterns of bone cement nanocomposites
with 1 wt% of LDH having different Mg : Al molar ratios. The
xed and optimum weight percentage of LDH (1 wt%) has been
taken from our previous work.29 The diffraction peaks at 5.96
,
5.85
and 5.78
of (001) planes for 2 : 1, 3 : 1 and 4 : 1 LDHs,
respectively, corresponding to the layer spacings of 1.48, 1.51,
and 1.53 nm, disappear in the nanocomposites mainly because
of the exfoliated/disordered structure of LDHs in the bone
cement matrix. The poly(methyl methacrylate) (PMMA) chains
present in the bone cement enter into the interlayer spacing of
the nanoLDH due to strong interaction (secondary forces)
between PMMA and nanoLDH causing this disordered layer
structure of LDHs in nanocomposites. Pure PMMA usually
creates highly intercalated nanocomposites with layered sili-
cate, where higher reections are also quite strong,38 while in
this study a disordered structure is expected because of superior
interaction, resulting in the disappearance of the XRD peak in
the nanocomposites. However, the high resolution TEM image
of the nanocomposite (4 : 1 LDH) clearly indicates the interca-
lated pattern (Fig. 2b) and the high magnication image shows
the individual fringe/layer distinctly with an interlayer spacing
of 1.65 nm, as compared to 1.53 nm in pure 4 : 1 LDH
(measured through XRD). Huang and William, showed the
nanostructure of the PMMA nanocomposite prepared through
suspension polymerization in organically modied layered
silicate either in an intercalated or exfoliated form from the
XRD patterns depending on the type of organic modier/nature
of interactions.38 The extent of polymer intercalation was more
due to functionalities in the organic modier and initial layer
spacing, as the threshold value required should be higher than
the radius of gyration of polymer chains. In this study, the lower
d-spacing of unmodied LDH restricts the insertion of PMMA
chains only to a certain extent causing an average spacing of
1.65 nm, as measured from the TEM image, while the XRD
patterns clearly indicate the exfoliated/disordered structure of
the LDH nanocomposites. It is worth mentioning that the
intergallery spacing in the nanocomposite even decreases as
compared to the organically modied layered silicate clay where
photopolymerization occurs inside the gallery, the organic
modier being the intercalant as well as the photoinitiator.39
Therefore, LDH nanocomposites have a great possibility to
enhance the mechanical and other properties of bone cement
due to insertion of polymer chains inside the gallery and good
dispersion of LDH in the bone cement matrix.
Structure
The structures of pure bone cement and its various nanoLDH
composites were examined through XRD. Bone cement shows
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Fig. 2 (a) X-ray diffraction patterns of 2 : 1, 3 : 1, and 4 : 1 nanoLDH and their respective bone cement nanocomposites (1 wt%) and (b) bright field high resolution
TEM image of the 4 : 1 LDH–bone cement nanocomposite.
diffraction peaks assigned to barium sulphate (matched with
JCPDS le) and an amorphous halo arising from PMMA in bone
cement. Fig. 3 compares the XRD patterns of the bone cement
and its 3 : 1 molar ratio of LDH nanocomposites. The diffrac-
tion peaks of BC-1 have been slightly shied towards the lower
angle region in Mg : Al  2 : 1 molar nanoLDH composite (ESI
Fig. S2†). Some major diffraction peaks of pure bone cement
(BC-0) appeared at 19.94
(020), 25.81
(021) and 32.76
(002)
and the respective diffraction peaks of BC-1 have shied to
19.9
, 25.76
and 32.71
, respectively.21 The shiing of diffrac-
tion peaks in BC-1 suggests incorporation of nanoLDH as a
defect into the inorganic lattice leading to distortion. In the case
of a higher molar ratio (Mg : Al  3 : 1), the diffraction peaks
remain the same, as compared to the pure bone cement
revealing no structural change of the nanocomposites. Similar
Fig. 3 XRD patterns of 0.5 and 1 wt% nanocomposites of LDH-3 : 1 and pure
bone cement (BC-0).
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results were also obtained in the remaining LDHs (4 : 1, 5 : 1
and 10 : 1 Mg : Al LDHs) nanocomposites.
Interactions
FTIR spectra of bone cement and its various LDH nano-
composites (1 wt%) are shown in Fig. 4. The transmittance peak
of the carbonyl stretching frequency (>C]O) has been observed
at 1732 nm in pure bone cement29 and this peak has been
shied to a lower wavenumber region to 1720, 1720, 1717, 1730
and 1730 cm1 for BC-2 : 1, BC-3 : 1, BC-4 : 1, BC-5 : 1 and BC-
10 : 1 nanocomposites, respectively. The maximum shiing of
the carbonyl peak was observed with the LDH molar ratio of the
4 : 1–bone cement composite. The shiing of the carbonyl peak
has been highlighted with a rectangular box and is clearly
shown in the inset picture. It is concluded from FTIR
Fig. 4 FTIR spectra of pure bone cement (BC-0) and its 1 wt.% nanocomposites
with various Mg : Al ratios, BC-2 : 1, BC-3 : 1, BC-4 : 1, BC-5 : 1 and BC-10 : 1.
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measurement that the 4 : 1 LDH experiences the maximum

interaction between polymer chains and nanoLDH while the
interaction was gradually lowered both in the higher and lower
molar ratio LDH for a xed composition (1 wt%) of LDH in the
nanocomposites. One new peak was observed in the nano-
composites at 451 cm1 and it represents the presence of
nanoLDH, otherwise the absence of the other LDH peaks is
mainly because of its low abundance.
The UV-Vis absorption spectra originated from electronic
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transitions between ground states to higher energy states,

particularly transitions from s, p and n to their respective
higher energy states of s*, p* and n*. UV-Vis spectra of the bone
cement and its various LDH nanocomposites are shown in
Fig. 5 in the wavelength range of 200–500 nm. The absorbance
peak of the n / s* transition is observed at 219 nm in pure
bone cement40 and another peak around 273–280 nm is due to
the p / p* transition which arises from unsaturated bonds
(mainly the >C]O group).41 In nanocomposites, the n / s*
peak position was almost intact as compared to pure bone
cement (BC-0), but the p / p* transitions have been shied
towards a higher wavelength region due to strong interaction
between the bone cement and nanoLDH. The peak shiing
occurs to 280, 282 and 284 nm for the 2 : 1, 3 : 1 and 4 : 1 LDH
nanocomposites against the pure bone cement peak at 277 nm
(inset gure). The red shi of about Dlmax ¼ 3–6 nm suggests
that nanoLDH withdraws the electron cloud from the carbonyl
group of PMMA.42 The peak position does not alter aer the 4 : 1
mole ratio of Mg : Al in LDH nanocomposites indicating a
maximum interaction for the 4 : 1 LDH nanocomposite. A
similar observation was found in FTIR studies. However, the
interaction between LDH and bone cement is quite strong as
evident from the peak shiing in FTIR and UV-Vis, as compared
to the pure bone cement.
Thermal behaviour
The thermal stability of a material can be measured when a
material is subjected to continuous heating and monitoring its
weight as a function of temperature in a thermogravimetric
analyser (TGA). The degradation temperature of a polymer is
Fig. 6 Plots of the TGA thermogrammes of BC-0, BC-2 : 1, BC-3 : 1, and BC-4 : 1
in inert nitrogen media and the inset figure shows the degradation temperature
zone corresponding to 5% weight loss of the materials.
taken at a temperature corresponding to 5% weight loss. Fig. 6
shows the weight loss behaviour of pure bone cement and its
various 1 wt% LDH nanocomposites as a function of tempera-
ture. The degradation temperatures of the pure bone cement
(BC-0), BC-2 : 1, BC-3 : 1 and BC-4 : 1 were found to be 235
,
247
, 260
and 270
C, respectively, (inset gure) indicating
better thermal stability of the nanocomposites as compared to
pure bone cement by creating a thermal barrier in the presence
of inorganic layered LDH homogeneously dispersed in the bone
cement matrix. Further, it is concluded that the thermal barrier
is enhanced with an increasing molar ratio of Mg : Al,
presumably due to greater interaction which causes better
dispersion of LDH in the matrix to act as a superior thermal
barrier. An approximately, 20
C increment in degradation
temperature is reported in the literature43 with organically
modied LDH disperse in PMMA and the much higher
improvement (35
C) in degradation temperature/thermal
stability at a higher Mg : Al mole ratio in this study is due to
greater interactions, which systematically increase up to the
Mg : Al  4 : 1 ratio. The degradation behaviour of bone cement
and its nanocomposites proceeds via two steps, consistent with
previous studies,44 indicating a similar thermal degradation

Fig. 7 DSC thermograms of pure bone cement (BC-0) and other indicated
Fig. 5 UV-Vis spectra of pure bone cement (BC-0) and nanocomposites with nanocomposites (BC-2 : 1, BC-3 : 1, and BC-4 : 1) and vertical lines represent the
Mg : Al ratio of 2 : 1, 3 : 1, 4 : 1, 5 : 1 and 10 : 1 LDHs (each with 1 wt % of LDH). Tg value of the indicated specimens.

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mechanism of the nanocomposites in the presence of
nanoLDH.
Fig. 7 shows the DSC patterns of bone cement and its LDH
nanocomposites. The glass transition temperatures (Tg) of BC-0,
BC-2 : 1, BC-3 : 1 and BC-4 : 1 have been found to be 98
, 100
,
104
and 108
C, respectively, showing higher transition
temperature as compared to pure bone cement mainly because
of restriction in segmental movement in presence of layered
LDH and intercalation. Moreover, the higher Tg for higher
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Mg : Al ratio LDH nanocomposites is envisaged from the greater
intercalation, as envisaged from Fig. 4 and 5, leading to
enhanced restriction in segmental movement. Similar incre-
ment of glass transition (10
C) has been reported with 1%
LDH in PMMA matrix.45 Thus, the thermal stability and glass
transition temperature increase in presence of nanoLDH and
further enhancement takes place with increasing the Mg : Al
mole ratio of the said properties because of greater interaction
and intercalation.
Mechanical behaviour
Fig. 8a shows the stress–strain curves of the bone cement and
its 1 wt% nanocomposites with varying Mg : Al mole ratios.
Nanocomposites exhibit better stiffness (modulus) as compared
to pure bone cement (Fig. 8b). Interestingly, the elongation at
break increases gradually with an increasing Mg : Al mole ratio
up to 4 : 1 followed by a systematic decrease with further
increasing Mg : Al ratios. Toughness as measured from the area
under the stress–strain curve is enhanced signicantly in the
nanocomposites and more than 100% increment occurs in the
BC-4 : 1. Mechanical properties of the bone cement and
nanoLDH lled composites are presented in Table 1 with the
record of percent improvements/changes. The stiffness of the
bone cement is higher than that of cancellous bone (100–
300 MPa)46 while that of the nanoLDH composite of bone
cement is signicantly higher than most of the bones. More-
over, the toughness of bone cement improved by about 12, 78,
107, 58 and 3% in BC-2 : 1, BC-3 : 1, BC-4 : 1, BC-5 : 1 and BC-
10 : 1, respectively, as compared to pure bone cement. The
comparative stiffness and toughness are shown in Fig. 8b. The
tensile strength also exhibits a similar trend and gradually
Fig. 8 (a) Stress–strain behaviour of bone cement and its indicated nanocomposites and (b) comparative bar diagrams of modulus and toughness of bone cement and
its indicated nanocomposites.
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Journal of Materials Chemistry B
increases up to a 4 : 1 mole ratio and shows a decrease with
further increase in the mole ratio of Mg : Al in LDHs. The higher
trend of toughness up to the 4 : 1 ratio is presumably due to
greater interaction, as reported above between the PMMA and
nanoLDH which suppress the crack growth mechanism by LDH
orientation towards the stress eld being strongly attached to
the polymer chains.47 Similar improvement with functionalized
nanostructured titania (1 wt%) embedded in PMMA based bone
cement has been reported earlier with considerable improve-
ment in fracture toughness (63%) and exural strength (20%) as
compared to the control because of the bifunctional monomer
used for surface treatment.48 Organically modied LDHs
considerably reduce the overall mechanical properties (tensile
strength and elongation at break) of pure PMMA43 while
signicant development in the all round mechanical properties
up to the 4 : 1 ratio in this study is unique and it is presumably
due to greater interaction at a high mole ratio. Moreover,
nanocomposites with a 4 : 1 molar ratio of Mg : Al in nanoLDH
are ideal for enhancing the mechanical properties of a bone
cement, both in terms of stiffness and toughness, which can be
applied for prosthetic implants. It is worth mentioning that
elongation at break and tensile strength rather decrease using
similar LDH (2 : 1 mole ratio with 1 wt% ller) dispersed in
polyethylene49 matrix, this might be due to poor interactions
between the polymer matrix and ller emphasizing the impor-
tant role of the interaction between the two phase materials, the
primary criteria for the energy dissipation toughening mecha-
nism.47 Hence, nanoLDH dramatically improves the overall
mechanical properties especially with a 4 : 1 mole of Mg : Al
composition of LDH because of the signicant interaction.
Hemolysis assay
Direct contact of any foreign material (implant) with blood may
create hemolysis and the permissible limit of hemolysis for a
biomedical implant material should be less than 5%.50 The
extent of hemolysis caused by an implant material actually
reects the bio-incompatibility of that biological implant.51 Due
to this problem, any implant material causing a high percentage
of hemolysis may show deleterious effects on patients. Fig. 9
shows the hemolysis percentage of ACD blood with heparinised
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Table 1 Mechanical properties of bone cement and its 1 wt% LDH nanocomposites at indicated Mg : Al ratios

% Increase Toughness % Increase Ultimate strength % Increase

Sample Modulus (GPa) in modulus (kJ m3) in toughness (UTS) (MPa) in UTS

BC-0 1.68 — 741.3 — 39.1 —

BC-2 : 1 1.84 8.6 835.2 12.66 46.9 19.94
BC-3 : 1 2.03 20.83 1319 77.93 56.9 45.52
BC-4 : 1 2.23 32.73 1534 106.93 59.6 52.42
BC-5 : 1 2.27 35.12 1175 58.50 60.1 53.70
BC-10 : 1 2.26 34.52 764 3.06 51.56 31.86
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Fig. 9 The bar diagram showing the percentage hemolysis of bone cement and
its different LDH nanocomposites (1 wt% LDH) with indicated molar ratios.

lms of pure bone cement and its different nanocomposites.
The percentages of hemolysis were found to be 3.6, 1.3 (P #
0.001), 1.9(P # 0.001), 2.1 (P # 0.01) and 2.9% for BC-0, BC-2 : 1,
BC-3 : 1, BC-4 : 1 and BC-5 : 1, respectively. Commercial bone
cement (BC-0) was reported to be biocompatible as its hemo-
lysis percentage was 3.6% (less than the limiting value of 5%).
Interestingly, nanocomposites show even lower hemolysis
exhibiting better biocompatibility as compared to pure bone
cement. Further, the slight increase in hemolysis with Mg : Al
ratio might be due to free interlayer water molecules in a higher
Mg : Al ratio LDH as observed in XRD and FTIR measurement
(Fig. 1a and b) since pure water acts as a hemolytic agent for
blood.52 On the other hand, strongly hydrogen bonded inter-
layered water molecules in higher Mg substitution with Al or
lower Mg : Al ratio keeps the hemolysis lower. However, the
percentage hemolysis in nanocomposites is less than pure bone
cement for every composition of LDH and it could be concluded
that blood compatibility is improved in the presence of
nanoLDH in nanocomposites.
Fig. 10 MG-63 cell adhesion on bone cement and its indicated nanocomposites
in terms of optical density. The absorption value was taken at 570 nm
wavelength.
respectively. Here LDH nanocomposites showed much greater
cell adhesion (optical density) with respect to the control which
was pure bone cement. Cell adhesion was almost constant for
BC-2 : 1, BC-3 : 1 and BC-4 : 1 and then decreased with an
increasing Mg/Al molar ratio in LDH. Greater cell adhesion in
nanocomposites up to 4 : 1 Mg : Al ratio vis-à-vis the control or
pure bone cement indicates the better biocompatiblity of the
bone cement nanocomposites with nanoLDH.
Cell viability
MG-63 cell viability on bone cement and its different molar
ratios of LDH nanocomposites has been examined through

Cell adhesion
The in vitro osteoconductivity of bone cement and its nano-
composites have been evaluated in terms of cell adhesion and
cell proliferation by using human osteoblast like cells (MG-63),
which produce phenotypic osteoblast markers and growth
factors.53 The adhesion of these cells on the specimens of bone
cement and its various LDH nanocomposites are shown in
Fig. 10. The optical density of the cells seeded on pure bone
cement (BC-0) was taken as a control. The optical density was
0.54, 1.26, 1.25, 1.16 and 0.6 in pure bone cement (BC-0), BC- Fig. 11 MG-63 cell viability of bone cement and its indicated nanocomposites
2 : 1(P # 0.01), BC-3 : 1(P # 0.01), BC-4 : 1(P # 0.01) and BC-5 : 1, with time intervals of 1, 3 and 5 days.

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MTT assay at the time intervals of 1, 3 and 5 days and is shown
in Fig. 11. Viability of the cells seeded on the tissue culture
surface was taken as a control. The cell viability was found to be
80% for pure bone cement aer 1 day, while it increased by
another 68% (P # 0.001), 45% (P # 0.05), 36% and 2% for BC-
2 : 1, BC-3 : 1, BC-4 : 1 and BC-5 : 1, respectively. Similarly aer
3 days, the viability was 84% in pure bone cement and
increased further by 58% (P # 0.001), 42% (P # 0.05), 37% (P #
0.05) and 10% for BC-2 : 1, BC-3 : 1, BC-4 : 1 and BC-5 : 1,
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respectively. The same trend was observed aer 5 days treat-
ment as well. In summary, the cell viability was found to be
signicantly higher in nanocomposites in comparison to pure
bone cement. However, the absolute value of cell proliferation
among the composites decreased with an increase in Mg : Al
molar ratio. Even though Mg metal is well known to be
biocompatible for biomedical applications,54 the biocompati-
bility, as observed from cell sustainability, steadily decreases
with its increasing concentration of Mg.55,56 With gradual
replacement of Mg with Al in a lower Mg : Al mole ratio LDH
exhibits better biocompatibility. Fig. 12 shows the uorescence
image of the MG-63 cell proliferation in all specimens in
support of the MTT assay. The live cell density per square cen-
timetre of BC-0, BC-2 : 1, BC-3 : 1, BC-4 : 1, BC-5 : 1 and control
was approximately 12, 25, 22, 24, 21 and 30, respectively, indi-
cating higher cell density for the LDH nanocomposites. The
better cell adhesion and cell viability of LDH nanocomposites as
compared to pure bone cement is also explained from their
better hydrophilic nature, as measured from the reduction in
contact angle of the LDH nanocomposites against pure bone
cement. The contact angles are 81
, 79
, 78
, 76.6
and 76.4
for
BC-0, BC-2 : 1, BC-3 : 1, BC-4 : 1, BC-5 : 1, respectively, since the
bioactivity of a material mainly depends on their hydrophilic
nature as it promotes the cell adhesion and proliferation. A
reduction in cell viability was observed in functionalized CNT
nanocomposites as compared to the control PMMA based bone
cement, even though the fatigue behaviour improved suffi-
ciently.57 Pure bone cement has the ability to induce expression
of osteoblast differentiation factor which can cause
Fig. 12 Fluorescence microscopic image of cells cultured on bone cement and the indicated nanocomposites after 1 day of cell proliferation.
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development of an osteolytic area at the bone-cement inter-
face.58 The presence of nanoLDH in bone cement has given
improved osteoconductivity (bioactivity) and it was established
through, enhanced performance of cell adhesion, proliferation
studies and greater cell viability in the presence of LDH, de-
nitely demonstrating a better biocompatible material of the
nanocomposites, or in this case as a grouting material.
Fatigue testing
In the previous section, the superior mechanical, thermal
properties and biocompatibility of the nanocomposites over
pure bone cement were demonstrated. In the present investi-
gation, nanocomposites have been used for grouting material
for real bone implant and studies have been conducted on the
versatile properties over pure cement. Comparative fatigue
resistance (life) of nanocomposites with respect to commercial
bone cement has been performed on specially designed speci-
mens, which mimic human hip prosthesis, as shown in the ESI
Fig. S1.† For each sample, the two best metallic stem implanted
femur specimens were selected for fatigue testing. Fatigue tests
were conducted at differently applied compressive loads from
1 kN to higher levels at a cyclic frequency of 3 Hz. The speci-
mens were subjected to 50 000 cycles at each increased level of
applied compressive load. The results of the high cycle fatigue
test of bone cement and 1 wt% nanocomposites are presented
in Fig. 13, wherein the number of cycles is plotted against the
applied load. It is obvious from the gure that the nano-
composite lled joint sustained an applied compressive load
cycle of 50 000 at each load level starting from 1 kN to 3.5 kN
and the Mg : Al  2 : 1 nanocomposite failed at 4 kN aer 6800
cycles and the Mg : Al  3 : 1 nanocomposite at 40 000 cycles
against the pure bone cement which failed at 10 000 cycles at a
lower load of 3.5 kN. On the other hand, the implant with 4 : 1
LDH nanocomposite sustained a compressive load of 4 kN and
failed at the higher load of 4.5 kN at 21 000 cycles. Thus, it is
clear from these results that the bone cement nanocomposites
have higher fatigue resistance under cyclic loading as compared
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Fig. 13 Bar diagram showing the failure points in terms of number of cycles for
pure bone cement and its 1 wt% nanocomposites with respect to varying applied
loads indicated on the X-axis.

to the pure bone cement and the fatigue resistance gradually

increases with the Mg : Al ratio in nanoLDH. Hence, both steady Fig. 14 The rabbit tibia X-ray images at the indicated time intervals of 0, 24 and
and dynamic measurements indicate a greater mechanical 40 days of the control (blank), BC-0 (pure bone cement) and BC-2 : 1 (nano-
composite). The arrows indicate the position of the hole during the course of time.
strength of nanocomposites vis-à-vis pure bone cement. Thus, it
may be concluded that nanocomposites of bone cement with
nanoLDH are denitely a better option to replace the
deposition takes place at the edges of the hole. The bone holes
commercial bone cement in total joint replacement for longer
lled with pure bone cement were almost the same up to 24
survival. Moreover, the improvement in fatigue behaviour or
days, but on the 40th day X-ray examination shows stable xa-
toughness with increasing the mole ratio of Mg : Al is primarily
tion of the bone using bone cement, calcium deposition on the
due to enhanced interactions as described in the above section.
edges (white colour) but yet to structure the new bone forma-
It is worth mentioning that the toughness of the bone cement–
tion. The bone healing takes place in 60 days using bone
MMT nanocomposite was increased only by 30%21 while a
cement as a lling agent. Interestingly, the rabbit bone hole
similar quantity (1 wt%) of nanoLDH enhanced more than
lled with bone cement nanocomposite has fully covered the
100% in toughness as compared to pure bone cement exhibit-
surroundings of the nanocomposite with calcium within
ing the superior properties of nanoLDH over nanoclay,
24 days post surgery. Aer 60 days post surgery, X-ray exami-
presumably due to greater interaction between the nanoLDH
nation shows the maximum calcium deposition (healing) was
and bone cement (PMMA), as revealed through greater shiing
observed in commercial bone cement, but in the case of the
in FTIR peaks, higher degradation temperature and the
control the healing process takes around 80 days time. From
enhanced extent of intercalation in the LDH–bone cement vis-
these results it is attributed that the bone healing was on a
à-vis the nanoclay–bone cement composite. From the combined
natural path in the control, while the relatively biologically inert
effect of mechanical, thermal and biocompatibility, the
commercial bone cement has quickened the bone growth to
nanoLDH composite of bone cement with a 4 : 1 mole ratio of
some extent, but the bone cement nanocomposite was proven to
Mg : Al is considered to be best as the biocompatibility is almost
be highly stimulating towards calcium deposition at the wound
similar to the 2 : 1 mole ratio of Mg : Al, but it has much
site i.e. it greatly favours the bone growth and cell adhesion.
superior steady and dynamic mechanical strength.
Hence, it is concluded that the bone cement nanocomposite is
more highly bioactive than the pure bone cement, which was
In vivo evaluation through radiography well tested in the in vitro cell culture examination and hemolysis
assay studies, helping in advancing the bone healing as
Radiography (X-ray imaging) is a primary and consistent diag-
compared to the control or using conventional bone cement as a
nostic technique to examine the pre- and post-surgery situation
grouting material.
of patients in orthopaedics, particularly bone fractures. The
X-ray images of rabbit tibia holes lled with pure bone cement,
nanocomposite and control (without lling) with different time Conclusions
intervals are shown in Fig. 14. The X-ray images were taken
every 5 days from the day post surgery. In the control, the bone Layered double hydroxides with a wide range of Al substitution
hole remains almost the same even aer 60 days, but calcium have been synthesized. The gallery spacing increases at higher

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Mg : Al mole ratios for pure LDHs and their nanocomposites
with PMMA based bone cement have been prepared without
using any solvent for its biomedical use. The polymer interca-
lates into the layers of the LDH making them disordered, but
stacked enough, as evident from there being no diffraction peak
in the XRD with layered patterns in the TEM bright eld image.
Both stiffness and toughness of the nanocomposites increase
without any trade off showing signicant improvement in the
mechanical properties aer Al substitution. The degradation
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temperature of the nanocomposites is enhanced up to 30
C as
compared to pure bone cement. The improvement in the
mechanical and thermal properties is explained on the basis of
greater interaction between PMMA and nanoLDH, as revealed
through FTIR and UV-Visible spectroscopy. The superior
bioactivity and biocompatibility of the nanocomposites over
pure bone cement has been veried through cell adhesion, cell
proliferation, hemolysis assay, and cell viability examined
through MTT assay. Mechanically stronger, thermally stable
and better biocompatible nanocomposites have been demon-
strated as a grouting material in bone implants exhibiting much
higher fatigue resistance showing a systematic increasing
tendency at higher Mg : Al mole ratios, as predicted from other
results. In vivo experiments on rabbits clearly demonstrate the
higher efficiency as a healing agent of the bone cement nano-
composite over pure bone cement and the control.
Acknowledgements
The author (Govinda Kapusetti) gratefully acknowledges nan-
cial support from the Council of Scientic and Industrial
Research (CSIR), Government of India in the form of fellowship.
The authors are thankful to Prof. S. C. Goel of the Department of
Orthopedics, IMS, BHU, for his interest in the work. The
authors also acknowledge the kind support of Dr D.K. Avasthi,
Mr Pawan K. Kulriya of IUAC, New Delhi for XRD measure-
ments. I am very grateful to my colleagues Mr Yogendra and Mr
Sudhakar for their intellectual support.
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