Applied Surface Science 257 (2010) 15331539

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Applied Surface Science

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In vitro bioactivity and biocompatibility of calcium phosphate cements using

Hydroxy-propyl-methyl-Cellulose (HPMC)
M. Anirban Jyoti a , Van Viet Thai b , Young Ki Min a , Byong-Taek Lee b , Ho-Yeon Song a,
a
Department of Immunology, School of Medicine, Soonchunhyang University, Cheonan, Chungnum 330-090, South Korea
b
Department of Biomedical Engineering & Materials, School of Medicine, Soonchunhyang University, Cheonan, Chungnum 330-090, South Korea

a r t i c l e i n f o a b s t r a c t

Article history: In this study, the bioactivity and biocompatibility of new calcium phosphate bone cements (CPC) using
Received 18 April 2010 Hydroxy-propyl-methyl-Cellulose (HPMC) was evaluated to understand the effect of HPMC on bone-
Received in revised form 15 August 2010 bonding apatite formation and biocompatibility. In vitro bioactivity was investigated by incubating the
Accepted 24 August 2010
CPC samples containing different ratios of HPMC (0%, 2% and 4% HPMC) in simulated body uid (SBF) for
Available online 15 September 2010
2, 7, 14 and 28 days. The formation of bone like apatite was conrmed on CPC surfaces by SEM and XRD
analysis. Higher HPMC content of CPC showed faster apatite deposition in SBF. A high Ca ion dissolution
Keywords:
prole was also reported with an increase of pH in all samples in SBF. The apatite formation ability of
Bioactivity
Biocompatibility
these CPC samples was found to be dependent on both surface chemistry and immersion time in SBF.
HPMC The In vitro cytotoxicity test showed that the CPC samples with 4% HPMC were fairly cytocompatible
CPC for broblast L-929 cells. SEM images showed that MG-63 cells were successfully attached to the CPC
SBF samples and well proliferated.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Bone defects by trauma, tumor or infection causes severe com-
plications in patients receiving orthopedic surgery due to the lack
of proper implantable materials. For the last few decades calcium
orthophosphate (CaP) compounds have been examined as potential
bone repair materials [1]. Many CaP compounds have been shown
to be chemically similar to natural bone tissues and they possess
both biocompatibility and bioactivity. Due to these properties, CaP
compounds are in high demand by biomaterial scientists [2]. Sev-
eral previous studies [36] have reported on the osteoconduction
of these biomimetic CaP compounds.
In the mid 1980s, Brown and Chow formulated a new calcium
phosphate cement, referred to as CPC [7]. The CPC powder pro-
vided a signicant step forward in the eld of calcium phosphate
biomaterial research and holds promise for use in the treatment of
bone defects. Ginbera et al. summarized the advantages of calcium
phosphate cements (CPC) over CaP ceramics: CPCs have a self set-
ting ability, which shortens the surgical time effectively; they have
improved injectability, which minimizes the need for aggressive
conventional surgical techniques; they can be molded to t per-
fectly on the implant site; and lastly, they have increased reactivity
and potentiality for use in controlled drug delivery [8]. The oste-
Corresponding author. Tel.: +82 41 570 2412; fax: +82 11 9421 2412.
E-mail address: songmic@sch.ac.kr (H.-Y. Song).
conductivity and bone regeneration of CPC has also been reported
to be excellent [9]. Since the initial development of CPC by Brown
and Chow, several compositions have been formulated with the aim
of improving its overall biocompatibility as well as its mechanical
properties [10].
The apatite formation ability of CPC has been studied and it
was shown that calcium phosphate cements can grow apatite layer
in simulated body uid (SBF) [11,12]. This apatite formation abil-
ity might vary depending on chemical formulation of different
CPC samples. Several studies also have been performed to under-
stand the mechanisms of apatite formation in SBF immersion.
But there is a scarcity in the data for the apatite formation abil-
ity of different CPC samples. Especially concerning the fact that
vast chemistry has been employed to manipulate more accept-
able formulations, but not the apatite formation mechanism. In
1991, Kokubo et al. proposed that bone like apatite formation
on the surface of an articial material is an essential require-
ment for that material to bond to living bones; thus, this property
is necessary for successful implantation in living body [13,14].
By using SBF one can easily reproduce this in vivo apatite for-
mation. In other words, in vitro apatite formation in SBF can be
used as a tool for understanding the in vivo nature of that mate-
rial, i.e. whether it is bioactive or not [13,14]. Moreover, some
investigators used SBF as a tool to synthesize apatite-like pow-
ders in vitro [15]. Several other studies reported that bioactive
and biocompatible materials form bone like apatite on surfaces,
which facilitated biodegradation, bioinduction of the materials

0169-4332/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsusc.2010.08.091
1534 M.A. Jyoti et al. / Applied Surface Science 257 (2010) 15331539
Table 1
Composition and mechanical properties (e.g. compressive strength, injectability) of 0%, 2% and 4% HPMC.
Sample name Powder components [wt%] Liquid components [wt%]
TTCPDCPD CSD Chitosan Citric acid
0% HPMC 80 20 2 20
2% HPMC 80 20 2 20
4% HPMC 80 20 2 20
or enhance bone like mechanical properties upon implantation
[16,17].
In our previous work, we fabricated and characterized the
mechanical properties of a new CPC injectable bone substitute (IBS),
which consisted of TTCP (tetra calcium phosphate), DCPA (dical-
cium phosphate anhydrous) and CSD (calcium sulphate dihydrate)
as the solid phase and chitosan, HPMC (Hydroxy-propyl-methyl-
Cellulose) and citric acid as the liquid phase [18]. Chitosan is already
known to be biocomplatible, biodegradable and osteoconductive by
several studies [19,20]. A more recent study showed that the CPC-
chitosan scaffold possessed exural strength nearly 50% to 3-fold
higher than the FDA-approved CPC control. This CPC-chitosan scaf-
fold also supported messenchymal stem cell (MSC) attachment and
proliferation [21]. Incorporation of gelling agent has been reported
to improve its injectability. In a previous study, the effect of HPMC
(hydroxypropyl methylcellulose) was measured and found to be
improved dramatically [22]. In addition, HPMC is the synthetic
hydrogel as well as naturally derived cellulose; this non-ionic cel-
lulose ether is widely used in pharmacology for controlled drug
release system [2325]. Daculsi and coworkers successfully used
the rst generation viscous HPMC solution associated with calcium
phosphate granules [23,24]. Citric acid (C6 H8 O7 ) plays an important
role as intermediate product of the Krebs cycle in human. Besides
it forms chitosanorganic acid complex [26]. It was found that
chitosan/hydroxyapatite composites gained enhanced mechanical
properties upon addition of a small amount of citric acid [27]. Liu
et al. in a study demonstrated that the concentration of citric acid
effected the setting times, mechanical properties and biocompat-
ibility of the cement composites consisting of calcium phosphate
and chitosan [28].
In this study, we will report on the in vitro bioactivity and bio-
compatibility behavior of newly fabricated CPC injectable bone
substitutes. The HPMC content was varied to understand the effect
of HPMC on apatite formation ability on the surfaces CPC samples
in SBF and general biocompatibility in vitro.
2. Materials and methods
2.1. Preparation of cement
The sample preparation was described in Ref. [18]. In brief, the
CPC bone substitute was prepared by mixing both the liquid phase
and solid phase together. The liquid phase was an aqueous solu-
tion of chitosan, HPMC and citric acid. The mass fraction of chitosan
(Sigma, Iceland; >85% deactylation degree) was 2 wt.% and the mass
proportion of citric acid (Samchun Pure Chemical Co. Ltd., Pyong-
taek, South of Korea) was 20%, 30%, and 40%, respectively. HPMC
was purchased from Sigma Aldrich. To understand the effect of
HPMC on bioactivity and biocompatibility, we varied the mass%
of HPMC in distilled water. The following HPMC concentrations
were used: 0%, 2% and 4%. The samples were named accordingly,
0% HPMC, 2% HPMC and 4% HPMC.
The powder component consisted of a mixture of TTCP, DCPD
(Sigma, USA), and CSD (Samchun, South of Korea). The TTCP pow-
der was synthesized using a solid-state method [19,20]. 73.69 wt.%
of TTCP-like and 26.31 wt.% of DCPD were mixed in a ball grinder
for 8 h in equimolar amounts to obtain the CPC powder. The CSD
Compressive strength (Mpa) Initial setting time (min)
HPMC 7 days 14 days
0 10.7 12.24 50
2 14.69 17.94 41
4 17.65 20.87 35
constituent was xed in the CPC powder at 20 wt% concentration.
The powder and liquid were mixed manually with a spatula for
45 s or around 1 min at a ratio of 1.8:1 [wt./wt.]. After mixing
until a chewing-gum-like slurry was always achieved, the slurry
was molded into tubes that were open at both ends (9.5 mm
in diameter 2 mm in length). Table 1 shows the composition
and mechanical properties of 0%, 2% and 4% HPMC samples. The
compressive strengths (measured by using a computer controlled
universal testing machine (R&B, Korea)) and initial setting of 0%,
2% and 4% HPMC were variable according to percentage of HPMC
content.
2.2. Bioactivity experiments in simulated body uid (SBF)
To evaluate the bioactivity of the ceramics in vitro, 0%, 2% and 4%
HPMC cements were immersed in simulated body uid and incu-
bated in a CO2 incubator with 5% CO2 and 100% relative humidity
at 37 C for 2, 7, 14 and 28 days intervals. The SBF solutions were
prepared according to the methods described elsewhere by Kokubo
et al. [13]. The amount of SBF for each sample was determined by
Vs = Sa/10, where Vs = volume of solution, Sa = surface area of the
samples [13]. SBF solutions and the respective samples were col-
lected at the end of each incubation period. After incubation, the
SBF solutions were well preserved by refrigeration at 4 C and the
CPC samples were washed with distilled water rst and then dried
in air by using a lter paper. The samples were then preserved in
ambient temperature until further use.
2.2.1. Study of surface morphology after SBF
To assess the surface morphology by SEM after apatite forma-
tion, we incubated samples in SBF. SBF immersed and incubated
samples were periodically collected after nishing each incubation
time. We collected the samples aseptically and dried it in drier at
40 C. SBF solutions were refreshed everyday at the same time. The
surface morphology of the cement was observed under scanning
electron microscopy (SEM, JSM-635, JEOL, Tokyo, Japan). Prior to
acquiring SEM images of the sample surfaces, each sample was
mounted on aluminum specimen stubs with gold and coated with
SPI-module Sputter Coater at 7 mA for 20 min.
2.2.2. Peak analysis by X-ray diffraction
The X-ray diffraction proles (XRD, D/MAX-250, Rigaku, Tokyo,
Japan) of different cements after SBF incubation were recorded by
X-ray powder diffraction analysis at a scanning speed of 2 /min.
2.2.3. Measurement Ca and P ion concentration in SBF
The Ca and P ion concentrations in the in SBF solution after
incubating the CPC samples for 2, 7, 14 and 28 days were mea-
sured using the ICP methods (ELAN 6000 ICP-Mass Spectrometer,
PerkinElmer, USA).
2.2.4. Measurement of pH change in SBF
The pH of the samples after incubation in the SBF solution for
2, 7, 14 and 28 days was measured using an electrolyte-type pH
meter.
 M.A. Jyoti et al. / Applied Surface Science 257 (2010) 15331539
2.3. Cell culture
Two types of cell lines were used in this study. For the cytotox-
icity analysis, we used Human broblast-like cells, L-929 derived
from mouse broblast. L-929 cells were cultured with RPMI media.
To study cellular attachment and adherence to the samples, human
osteoblast-like cells, MG-63 derived from human osteosarcoma,
were used. Both RPMI and DMEM media were further supple-
mented with 10% fetal bovine serum (FBS, Hyclone), 1% antibiotics,
50 g/ml ascorbic acid (Sigma) and 10 mM -glycerophosphate
(Sigma) prior to subculture to facilitate growth and inhibit bac-
terial infections. Both the cell types were sub-cultured using falcon
T75 ask (BD Falcon) and incubated at 37 C, 95% humidity and 5%
CO2
2.4. Cytotoxicity assay
The cytotoxicity of each CPC samples was determined using the
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide) assay [29]. The cellular viability% against each of the
fabricated cements was determined with coherent dilutions of the
samples following the standard testing protocol (ISO 10993-5) [30].
In brief, RPMI medium was used as the diluent to obtain the sample
extract. The specimens were kept in a shaking incubator at 70 rpm
and 37 C for 72 h. After incubation, the extractions were collected
by ltration and serially diluted (100%, 50%, 25%, 12.5%). The diluted
extracts were then placed into a 24-well culture plate that was
previously sub-cultured with L-929 cells (1 104 cells/ml). The
24-well culture plate was then placed in a CO2 incubator and incu-
bated for 72 h at 37 C. After incubation, the MTT solution (100 l of
5 mg/ml) was added into the wells and the plate was re-incubated
at 37 C in 5% CO2 for 4 h. In the nal step, the supernatant was
removed from the plate and 1 ml of dimethyl sulfoxide (DMSO)
was added to each well. After mixing with gentle shaking, purple
fomazan crystal formed and dissolved. The solution was collected
and pipetted into a 96 well plate the absorbance was measured in
an ELISA reader. The absorbance was determined at a wavelength
of 595 nm. The cell viabilities were measured as the percentage of
optical densities of medium containing the extract solution to the
optical densities of the fresh medium.
2.5. Preparation of samples for analysis by SEM
Osteoblast-like cells, MG-63, were used to observe the attach-
ment behavior of the cells onto the CPC samples and the cellular
morphology of the attached cells. First, freshly sub-cultured cells
were counted and 2 105 cells were dropped by micropipette
onto each of the CPC surfaces (hence denoted as cell-CPC con-
struct). These cells were xed onto the surfaces by maintaining
the cell-CPC construct in 2% gluteraldehyde and 0.1 M phosphate
buffer (PB, pH 7.45) at 4 C for 1 h. After the xation stage was
complete, constructs were washed by PB buffer twice to remove
any unattached cells and debris. Samples were then dehydrated
using a series of ethanol (50%, 70%, 90%, 95% and 100%). Reagent
graded hexamethyldisialazane (HMDS, Sigma H4875) was used to
dry the CPC samples. Finally, the dried samples were imaged by
SEM (JSM-5410-V, Jeol) following carbon coating by SPI-module
Sputter Coater at 7 mA for 20 min.
3. Result and discussion
After Brown and Chows discovery of the new CPC powder
that possessed a self-hardening ability, calcium phosphate cements
became one of the most important and widely studied elds for
biomaterial scientists as these cements form hydroxyapatite and
are osteoconductive. Because the hardened form of the sintered
1535
hydroxyapatite faces cannot be directly t into surgical sites, they
need to be reshaped prior to surgery, which is time consuming and
also increase the risk ratio of trauma and bone lose [31]. More-
over, several studies have reported a lack of mechanical strength
and brittleness in ceramic HAp [7]. In our previous study, we fab-
ricated a new bone substitute that consisted of CPC powders and
HPMC and evaluated its mechanical properties [18]. In this previous
investigation, 04% HPMC was added to the composite to examine
the effect of HPMC on mechanical properties and bioactivity. The
newly fabricated CPC injectable bone substitute displayed satisfac-
tory mechanical strength, where the compressive strengths were
measured to be greater than 15 MPa (Table 1). In addition, the Initial
setting time was below 15 min. In this present work, we studied the
in vitro bioactivity and biocompatibility of 0%, 2% and 4% HPMC sam-
ples to understand and predict how these samples would behave
in vitro after implantation. Fig. 1 shows the typical fractured sur-
face morphology of 0%, 2% and 4% HPMC by SEM images. All the
three surfaces are rough with smooth dune like appendages that
seem to appear from the initial reaction between liquid and solid
phase orientation in the IBS. The degree of indigenous roughness
was increased in IBS samples by the increase of HPMC percentages.
This characteristic morphological change was closely investigated
in Fig. 1. Also it was found that almost all surfaces have irregular
shaped micro porous structure running through the inside dune
of IBS. Rectangular shaped, micron to submicron particles of solid
phase were also found on surfaces of each IBS (Fig. 1).
3.1. In vitro bioactivity
3.1.1. Apatite morphology
Samples containing 0%, 2% and 4% HPMC were incubated in SBF
solution for 2, 7, 14 and 28 days and then the surfaces of each sam-
ple were imaged by SEM. Fig. 2 shows the surface morphologies of
the samples after different incubation periods in the SBF solution.
These images clearly conrmed apatite formation. The volume of
the apatite was found to increase with an increase in the HPMC
percentage as well as with an increase in the incubation time. The
surfaces of 0% HPMC was irregular in shape with rough morphol-
ogy. Also, there were some big cracks and pores in the 0% HPMC
sample after 2 days of incubation. After 7 days of incubation, 0%
HPMC showed some apatite formation with some cracks and pores
on the surfaces. At longer incubation times (14 and 28 days), an
abundant and conuent apatite formation was observed on the sur-
faces of the 0% HPMC sample. A very similar pattern was observed
as the HPMC % was varied (Fig. 2). The amount of apatite forma-
tion increased as the percentage of HPMC added to the sample was
increased, i.e. the degree of apatite layer formation was as follows:
4% HPMC > 2% HPMC > 0% HPMC. The crystalline-like apatite was
needle shaped and ranged from 5 mm after an incubation period of
2 days to 0.5 mm after an incubation period of 28 days.
The in vitro bioactivity of a biomaterial can be easily predicted
by using simulated body uid (SBF), which possesses a similar
ionic concentration as human blood plasma and, thus, it can sim-
ulate the microenvironment of a bone inside the body [13]. Since
several studies have reported that calcium and phosphorous ion
nucleation onto bone surfaces facilitates bone formation and some
bioglasses form crystalline like bioactive apatite in SBF while other
non-bioactive materials do not, Kokubo et al. suggested that SBF
can be used as a tool for predicting in vitro bioactivity. Detailed
studies by Kokubo et al. on bone like apatite formation through SBF
incubation of various biomaterials suggest that this apatite might
bond tightly with surrounding bones [13,14]. Based on Kokubus
ndings and suggestion, we examined the apatite formation abil-
ity on different CPC injectable bone substitutes that vary in HPMC%
in this study. It was evident from the result shown in Fig. 1 that
this apatite formation ability is dependent on the surface morphol-
1536 M.A. Jyoti et al. / Applied Surface Science 257 (2010) 15331539

Fig. 1. Scanning electron microscopic images of three IBS; (a) 0% HPMC, (b) 2% HPMC and (c) 4% HPMC.

ogy and chemistry as well. As the HPMC content in the cement
was varied in this experiment to affect the surface morphology, it
was gured out that apatite formation could be affected by surface
morphology and thereby surface chemistry.
3.1.2. Peak analysis of the apatite
After forming the apatite, it was therefore important to check
the peaks of the apatite components that arose from SBF incuba-
tion. Fig. 3 shows the peaks of the different components (TTCP,
HAp, DCPD, and CSD) in samples after SBF incubation for 2, 7, 14
and 28 days. In the XRD prole, the 0% HPMC had several TTCP
peaks at 2 days after incubation into SBF. However, the TTCP peaks
gradually disappeared at longer incubations times, while several
HAp peak started to appear. In regards to HPMC%, the peaks fol-
lowed a very similar trend, i.e. several HAp peaks were obtained in
2% and 4% HPMC, which replaced the TTCP peaks of 0% HPMC. In
addition, the CSD peak remained unchanged throughout the incu-
bation period. Therefore it is assumed that TTCP and DCPD in the
CPC cement have effects on apatite formation in SBF depending on
two factors. One is the time of SBF incubation and another is the
HPMC content. As the HPMC content in the CPC also affects the sur-
face morphology, injectability and mechanical strength [18], in this
experiment it is suggested that addition of HPMC could be bene-
cial for both biological properties and bioactivity in a CPC injectable
bone substitute.
3.1.3. Change in CaP ion concentration and pH in SBF
Fig. 4(a and b) describes the calcium and phosphorous ionic con-
centration in SBF at 2, 7, 14 and 28 days. The Ca ion concentration
in SBF increased as the time of SBF immersion increased. Fig. 4(a)
actually describes the fast dissolution behavior of Ca ion from the
CPC samples into SBF. The highest dissolution rate of Ca ion was
observed in 2% HPMC. All of the CPC samples show a fast dissolu-
tion rate at days 2 and 7 followed by a relatively slow dissolution
at days 14 and 28. Especially in case of 2% HPMC, calcium ion con-
centration was found decreasing in SBF at day 28 from its peak
level of concentration in SBF. On the other hand, the concentra-
tion P ions in SBF were found to decrease in an abrupt manner
in SBF from the beginning of SBF incubation in all CPC samples
(Fig. 4b). The decreasing rate of phosphorous ion was faster in 4%
HPMC than the other two samples. At the beginning of SBF incu-
bation, the morphologically rough surfaces and open spaces of the
samples were virtually open for both dissolution and nucleation
of the ions. When the ca ions were being dissolved into the SBF,
Ca and P ions also began to deposit on the surfaces of CPC sam-
ples, reacted with CPC surfaces and helped form the apatite layer.

Fig. 2. SEM images of surfaces of IBS samples at different incubation periods in SBF. The samples (0%, 2% and 4% HPMC) showed crystalline apatite formation in various
amounts after incubating for 2, 7, 14 and 28 days in the SBF solutions.
 M.A. Jyoti et al. / Applied Surface Science 257 (2010) 15331539 1537

Fig. 4. Ca and P ion concentration in SBF, (a) Ca ion and (b) P ion at 2, 7, 14 and 28
days of incubation as a function of immersion time.

was attributed to the rough surface morphology of the sample. The

surfaces of specimens were irregularly shaped with large dune like
appearances and sub micron pores. This rough surface might cre-
Fig. 3. XRD proles of 0%, 2% and 4% HPMC samples after 2, 7, 14 and 28 days of ate a favorable microenvironment where calcium and phosphorous
incubation in SBF.
ion tended to concentrate and react with the surface to form the
apatite layer [33].
In this study, we carried out the SBF incubation of the CPC sam-
Initially there was a strong tendency of the ions to nucleate onto
ples in a closed vessel and in static condition. Previous studies
the surfaces quickly and, thus, they saturated the surface. While
suggest that CaP apatite growth in in vitro models is initiated by
monitoring the ion concentration during SBF incubation, the SBF
heterogeneous nucleation of the Ca and P ions [34]. Therefore mea-
was not changed everyday. So measuring the ion concentration as
a function of incubation period or time provided a measure of the
ionic concentration status in the SBF solution. But the change of pH
in SBF was not only time dependent but other factors inuenced
the pH of CPC immersed SBF solution. The pH of all of the cements
rapidly increased within two days of incubation followed by a slow
decrease (Fig. 5). The rapid increase of pH at day 2 was mainly
occurred by the high concentration of Ca ion into the SBF solutions
by dissolution. The starting pH of SBF was set to 7.43. At day 2, the
highest pH (pH 7.5) was observed in 0% HPMC due to the highest
dissolution of Ca ion in this solution. Although a high concentration
of Ca ion was also observed in later stage of SBF immersion, a fall
in pH in all samples was observed. Release of hydroxyl radical from
the sample surface chemistry might play a role in pH decrease in
this stage. In addition, the pH in later stage was assumed to change
due to mainly the acidic DCPD and citric acid content in the sample.
The liquid phase of the cement was acidic. Therefore an increase in
hydroxyl ion in SBF solution was believed to responsible for pH fall
at days 7, 14, and 28 (Fig. 4). However this type of ionic exchange
is thought to be blessing in case of calcium phosphate apatite for- Fig. 5. Changes of pH in SBF at 2, 7, 14 and 28 days of incubation as a function of
mation by electrostatic interaction [32]. Also the rapid nucleation immersion time.
1538 M.A. Jyoti et al. / Applied Surface Science 257 (2010) 15331539
Fig. 6. Fibroblastic cellular viability as assess by the MTT assay. Control cells cul-
tured without extract solution, and three other dilutions were used: L-929 cells
cultured with control (100% RPMI Media) 12.5%, 25%, 50% and 100% extract solution,
respectively.
suring the ionic concentration in SBF is a part of understanding
the mechanism of this nucleation. Changes in Ca and P ion con-
centration in SBF was measured by ICP method and illustrated in
Fig. 4. The calcium concentration in this study was found increas-
ing throughout the incubation period whereas the phosphorous ion
concentration was decreasing. It clearly indicates that calcium ions
are dissociated from the CPC samples and dissolved into SBF. On
the other hand, P ions tend to nucleate onto the surface. Initially
this type of result was not suspected. However, the apatite forma-
tion ability of each of the CPC injectable bone substitutes followed
the pattern as observed by other studies on titanium or bioglass
surfaces [35,36].
It is well known that SBF is supersaturated toward hydrox-
yapatite (HAp) and octacalcium phosphate (OCP). Therefore any
dissociation of ions will break this equilibrium condition and tend
to precipitate onto the surfaces [34]. The ICP result conrmed a
higher dissociation rate of Ca ion from the beginning of SBF incu-
bation. We assumed that release of hydroxyl ion also took place
along with cationic dissociation. On the other hand P ion was found
decreasing in SBF from the beginning of SBF incubation. The rapid
decrease in P ion in SBF can be explained in the vicinity of Ca ions
in SBF for apatite formation. CaP nanoparticles can be clustered
and aggregated by the inuence of supersaturation condition. These
aggregated clusters are negatively charged. Upon release of Ca and
OH ions in SBF solution, the surfaces of CPC IBS become positively
charged. At day 2 most of the sample started to produce apatite
onto their surfaces except 0% HPMC. From ICP result, it was shown
that 0% HPMC had lower phosphorous ion precipitation rate but
Fig. 7. SEM images of the cellular morphologies on 0%, 2% and 4% HPMC samples. Note: on 0% HPMC sample surface, cells are hardly expressing philopodia. On 2% HPMC, a
cell growth was fair. The maximum philopodial expression was observed in 4% HPMC.
higher calcium ion dissociation rate compared to other samples.
However the high pH at day 2 might affect the apatite formation in
2% HPMC in SBF. But as the immersion time increased, the needle
shaped crystalline apatite layers were found growing and increas-
ing. Also concerning the HPMC content in CPC samples, apatite
layers were found growing and increasing as observed in 2% and
4% HPMC. Therefore it was clear that apatite formation onto the
surfaces was dependent on time as well as surface chemistry.
3.2. Cellular viability
Apatite layer formation was also observed by Liu et al. where
they suggested that the apatite layer enhanced fast bone bonding
since they are very similar to that of natural bone [28]. In this study
we performed the cytocompatibility of 0%, 2% and 4% HPMC by cell
viability assay and SEM.
Fig. 6 showed the cytotoxic response of each of the 3 samples
as assessed by the semi automated MTT experiments. Fibroblastic
cells (L-929) were cultured with the extracted solution of each of
the four IBS cements for 72 h and then assayed for cell viability
relative to the control (100% medium). Based on the cytotoxicity
results, a fairly acceptable viability of the cells was observed on 2%
and 4% HPMC. But 0% HPMC showed a slightly lower viability (61%
at 100% extract solution). In other case, the general survival rate of
the cells for 2% and 4% HPMC samples was satisfactory being 74%
and 81% at 100% dilution (not using the fractions), respectively.
3.3. Cellular attachment and morphology on CPC samples
SEM images were used to verify cellular attachment and to char-
acterize the cellular morphology of the attached osteoblast cells
MG 63, as shown in Fig. 6ac. MG-63 cells were grown on top sur-
faces, incubated at 37 C and dried in an ethanol series (50100%)
and HMDS (hexamethyldisialazane). Cells were shown to be well
attached and proliferated to each of the surfaces of the CPC sam-
ples. All cells imaged by SEM were round in shape. Therefore, it was
assumed that cells were in the early stage of cellular proliferation.
Cell wall texture was rough and bleb-like vesicular protuberance
was evident. However, in comparison 4% HPMC showed the best
MG-63 cellular growth behavior on top of its surface (Fig. 7c). This
attachment was facilitated by several actin-like laments around
the cells. There was also some evidence of the presence of dense
materials around the cells. These were cytoplasmic extension with
extracllular matrix (ECM). The amount of lament expression was
dependent on the surface morphology and chemistry. More l-
aments were observed on 4% HPMC (Fig. 7c) than on 2% HPMC
(Fig. 7a). The cells were 6.57.0 mm in diameter on the 0% HPMC
and 7.08.0 mm in diameter on the 2% and 4% HPMC.
 M.A. Jyoti et al. / Applied Surface Science 257 (2010) 15331539
4. Conclusion
In this present study, we evaluated the bioactivity and bio-
compatibility of a CPC injectable bone substitute that consisted of
TTCP, DCPD and CSD in the powder phase with variable amounts of
HPMC (0%, 2% and 4%). Among the different liquid components,
HPMC was found to have the greatest effect on increasing the
strength of individual IBS. In vitro experiments showed the effect
of HPMC on bioactivity and biocompatibility. The in vitro study
indicated that the surfaces of the CPC injectable bone substitutes
were capable of nucleating bone like apatite, which has been previ-
ously shown to increase bioactivity. XRD prole showed that TTCP
and DCPD were converted into more stable HAp in SBF. However a
rise in calcium concentration in all samples was measured by ICP
method. Along with ICP data, pH change in SBF solution was also
observed. An electrostatic force was postulated for the apatite for-
mation in HPMC added CPC samples. Therefore we conclude that
apatite formation ability in these cements mainly depends on sur-
face chemistry of the CPC samples as well as immersion time in
SBF. Cellular toxicity levels were measured and a 81% viability was
observed in 4% HPMC samples. In addition, SEM imaging on MG-63
cells grown on CPC samples revealed that cells were successfully
attached to the surfaces. However, philopodial expression was only
observed in cells grown on 4% HPMC. Still further study is required
to demonstrate the bone bonding ability of these samples in animal
models.
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