Article

Journal of Biomaterials Applications

2014, Vol 28(5) 739–756
! The Author(s) 2013
Bioactive glass incorporation in calcium Reprints and permissions:
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phosphate cement-based injectable bone DOI: 10.1177/0885328213478256
jba.sagepub.com
substitute for improved in vitro
biocompatibility and in vivo bone
regeneration

Alexander Sadiasa1, Swapan Kumar Sarkar1, Rose Ann Franco1, Young Ki Min2 and
Byong Taek Lee1

Abstract
In this work, we fabricated injectable bone substitutes modified with the addition of bioactive glass powders synthesized
via ultrasonic energy-assisted hydrothermal method to the calcium phosphate-based bone cement to improve its bio-
compatibility. The injectable bone substitutes was initially composed of a powder component (tetracalcium phosphate,
dicalcium phosphate dihydrate and calcium sulfate dehydrate) and a liquid component (citric acid, chitosan and hydroxyl-
propyl-methyl-cellulose) upon which various concentrations of bioactive glass were added: 0%, 10%, 20% and 30%.
Setting time and compressive strength of the injectable bone substitutes were evaluated and observed to improve
with the increase of bioactive glass content. Surface morphologies were observed via scanning electron microscope
before and after submersion of the samples to simulated body fluid and increase in apatite formation was detected using
x-ray diffraction machine. In vitro biocompatibility of the injectable bone substitutes was observed to improve with the
addition of bioactive glass as the proliferation/adhesion behavior of cells on the material increased. Human gene markers
were successfully expressed using real time-polymerase chain reaction and the samples were found to promote cell
viability and be more biocompatible as the concentration of bioactive glass increases. In vivo biocompatibility of the
samples containing 0% and 30% bioactive glass were evaluated using Micro-CT and histological staining after 3 months of
implantation in male rabbits’ femurs. No inflammatory reaction was observed and significant bone formation was
promoted by the addition of bioactive glass to the injectable bone substitute system.

Keywords
Bone, bone regeneration, bioactive glass, biocompatibility, calcium phosphate cement

Introduction Studies by Brown and Chow in 1986 led to the for-

Administration of bone substitutes are often done via mulation of a new calcium phosphate cement (CPC)
graft reconstruction, which requires surgery that may composed of tetracalcium phosphate (TTCP) and dical-
cause infections, large muscle retraction and post- cium phosphate dihydrate (DCPD, CaHPO4-2H2O) or
operative pain. These reasons have led to the increasing
popularity of minimally invasive techniques and to the
development of injectable bone substitutes (IBSs). An 1
Department of Biomedical Engineering and Materials, School of
IBS, can be molded into the shape of a bone cavity and Medicine, Soonchunhyang University, Chungnam, South Korea
2
can harden on injection, thus eliminating the need of Department of Physiology, College of Medicine, Soonchunhyang
University, Chungnam South Korea
complicated surgery.1 These substitutes provide an
alternative for patients requiring bone reconstruction Corresponding author:
Byong Taek Lee, Department of Biomedical Science, Director, Institute of
when auto-grafting and other means of surgical inter- Tissue Regeneration, College of Medicine, Soonchunhyang University,
ventions may not be sufficient or pose more disadvan- 366-1 Sangyoung-dong, Cheonan City, Chungnam, 330-090, South Korea.
tages than advantages. Email: lbt@sch.ac.kr
740 Journal of Biomaterials Applications 28(5)

dicalcium phosphate anhydrous (DCPA, CaHPO4).2,3 since their discovery in 1969 because of their ability
The CPC led to great developments in IBS and was a to show both osteoconduction and osteoproduction.22
real breakthrough in the field of bioceramics, offering Bioactive glasses also display impressive bioactivity as
different properties such as self-setting ability, good they form a biologically active layer on their surfaces
injectability, perfect moldability and increased reactiv- when implanted. This characteristic is thought to play
ity and high feasibility in controlled drug delivery.3–6 an important role in the formation of direct bone
Various modifications were studied and applied to bonding.23–25
the CPC-based IBS system to improve its performance In this study, we report the ability of bioactive
in clinical applications.6,7 These modifications con- glasses to improve the biocompatibility of IBSs.
cerned further improvement in the material’s properties Bioactive glass materials of different concentrations
such as setting time and compressive strength as well as were added to CPC IBSs and the in vitro and in vivo
its biocompatibility. Addition of calcium sulfate dihy- biocompatibility behavior of the substitutes were
drate (gypsum, CSD, CaSO42H2O) to the CPC was observed. The effects of the bioactive glass concentra-
observed to have the ability to react with phosphate tions on the material and physical properties of the IBS
ions to form HAp crystals and improved its resorption were also investigated.
rate after the IBS system was implanted in vivo, making
the mixture of CSD and CPC a good framework for an
IBS system.8 Materials and methods
Several additives were also found to elevate and
Preparation of the IBS
enhance the chemical properties as well as the biocom-
patibility of an IBS system.6,7 In our previous study, the IBSs were prepared in accordance with the study of
liquid part of an IBS system consisted of a mixture of Thai et al.2 by combining two phases: a solid and a
chitosan, hydroxyl-propyl-methyl-cellulose (HPMC) liquid phase. The liquid phase consisted of 2% chitosan
and citric acid. These components were found to have (Sigma, Iceland; >85% deacetylation degree), 4%
different effects and elevated the performance of the HPMC (Sigma Aldrich) and 10% citric acid
IBS by improving its mechanical and biological proper- (Samchun Pure Chemical Co. Ltd., Pyongtaek, South
ties.2 HPMC, a gelling agent that can hydrogenate to of Korea), all mixed together to form an aqueous solu-
water and form a viscous solution, improves the cohe- tion. The solid phase of the sample was prepared by
siveness of CPC, enabling it to pass through a syringe combining TTCP, DCPD (Sigma, USA) and CSD
and be injected.9,10 Chitosan, a naturally derived poly- (Samchun, South of Korea). The TTCP powder was
mer, is a product of chitin hydrolysis and is widely used synthesized using a solid-state method.2,26 The molar
in biomaterial fabrication such as IBS to improve bio- fractions of DCPA and CaCO3 were set at 0.526:
compatibility.11 It resulted from the trend in implanta- 0.487. Such conditions were brought about by the
ble applications, in which nature-derived materials were investigation of Burguera et al.27 on the effects of the
desired for their potential to improve biomaterials in overall calcium to phosphate (Ca/P) ratio of TTCP syn-
terms of biocompatibility and promote faster heal- thesis on the existence of impurities. DCPA and CaCO3
ing.12–14 Citric acid, a diprotic acid, behaves as a lique- were ground using a planetary ball grinder for 6 h. Ca/P
fier admixture and improves the injectability of CPCs.15 ratios between 1.9 and 1.95 were regarded as the safe
It is an ingredient of human hard tissue and is believed approximations for the formulation of TTCP. TTCP,
to affect bone formation in humans by absorbing cal- 74 wt%, and DPCD, 26 wt%, were mixed in a ball grin-
cium ions during both reaction and production der for 8 h in equimolar amounts to obtain the CPC
phases.16 In several studies, the compressive strength powder. CSD was added in the IBS system at a fixed
of the IBS increases as the concentration of citric acid concentration of 20%.
increases.1,17,18 However, despite the improvement of Bioactive glass material was synthesized using ultra-
the material properties, the current IBS system still sonic energy-assisted hydrothermal method.28 The
needs to improve especially biocompatibility because powder was prepared by combining calcium nitrate
it was found out that citric acid delays apatite forma- (Samchun Pure Chemical, Korea), diammonium
tion.19 Citric acid may also induce an inflammatory hydrogen phosphate (Samchun Pure Chemicals,
response in vivo and according to Kurahina et al.,20 Korea) and sodium silicate solution (as 37% SiO2 in
TCP hardened by acid does not bind directly to the NaOH solution, Shwa, Japan). The ingredients were
bone of rabbits.18 mixed to produce bioactive glass material with precur-
Bioactive glasses are silicate glasses containing com- sor reactants of: 60% SiO2, 36% CaO and 4% P2O5
ponents like calcium and phosphate together with alkali with balanced Na2O. The resulting mixture was then
metal or rare earth metals.21 These materials have dissolved in de-ionized water and placed to an ultra-
attracted much interest in the field of bone grafting sonic bath for 4 h. The resulting powder was washed
Sadiasa et al. 741

with de-ionized water and filtered. It was further Korea) at a unilateral crosshead speed of 1 mm/min.
dried in 80 C oven for 24 h then calcined at a tempera- Compressive strengths were computed and plotted by
ture of 600 C. the Helio-X software. Reported values were the average
The bioactive glass materials of different concentra- values of 5 trials with standard deviations.
tions (0%, 10%, 20% and 30%) were added to the IBS
system and the resulting products were named accord-
ing to the sample’s bioactive glass material content:
In vitro experimentation
IBS-BG0, IBS-BG10, IBS-BG20 and IBS-BG30. Bioactivity experiments using simulated body fluid. The fabri-
After the preparation, the solid and liquid phases at cated samples were placed in a 12-well culture plate.
a ratio of 1.8:1were mixed manually with a spatula for Three sets of plates were prepared, each containing
1–2 min. Chewing gum-like slurry was achieved after the fabricated samples. Each sample was submerged
every mixing and was molded into cylinders and in simulated body fluid (SBF). This amount of fluid
tablet-like forms. to be placed in each sample was determined by the
Vs ¼ Sa/10, where Vs ¼ volume of the fluid (mL) and
Sa ¼ surface area of the sample (mm2).30 The samples
Physical and mechanical properties of the IBS were placed in an incubator set at 37 C temperature,
Characterization of the materials. The bioactive glass used 95% humidity and 5% CO2 for 1, 7 and 28 days with
in this paper was analyzed by attenuated reflectance the SBF solution changed every 24 h. After the
Fourier transform infrared spectroscopy (FTIR) incubation period, the samples were dried and analyzed
(Spectrum GX, PerkinElmer, USA). The infrared spec- for its XRD pattern while some parts were visualized
tra of the samples were measured over a wavelength using an SEM.
range of 600–2600 cm1, and were collected from 64
scans with a resolution of 4 cm1. Cell culture. MG63 cells, osteoblast-like cells, were used
The morphologies of the powders and samples after to study the biological capability of the samples to pro-
setting were observed using a scanning electron micro- mote cellular proliferation, attachment and adherence.
scope (SEM, JSM-635, JEOL, Tokyo, Japan) after 7 The cell line was obtained from the Korean Cell Line
days of incubation in 37 C atmosphere with 100% Bank and cultured using DMEM media. The culture
humidity. The samples were fixed in a sample holder medium was supplemented with 10% fetal bovine
and coated with platinum using SPI-module sputter serum (FBS, Hyclone) and 1% penicillin/ streptomycin
coater at 7 mA. antibiotics, prior to subculture, to facilitate cellular
The samples were analyzed for its x-ray diffraction growth and avoid contaminations, especially from
(XRD) pattern. The samples were pulverized and their microbial organisms. The cell cultures were plated in
profiles were obtained with an XRD machine (D/Max- cell culture plates (T75 flask, BD Falcon) and incubated
250, Rigaku, Tokyo, Japan). The machine was set to at 37 C, 5% CO2. At confluence, the cells were washed
emit CuKa radiation and generate 30 kV and 200 mA at with phosphate buffer solution (PBS) and collected
various diffraction angles of 20 to 60 at the angle rate using trypsin-EDTA. The collected cells were then sub-
of 2 /min. cultured in a new flask.

Setting time measurement. The samples were allowed to Cell proliferation examination. Proliferation patterns of the
set after mixing. The setting time of each sample was MG63 cells on the fabricated samples were observed
obtained by the Gilmore Needle Test according to using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazo-
international standard ISO 9917.29 The samples were lium bromide (MTT) assay method. Prior to the start of
considered set when a 400 g weight with a needle tip the experiment, the components of the samples were
of 1 mm in diameter no longer created an indentation prepared. The liquid component was sterilized via
on the surface of the cement. The procedure was con- ultrafiltration (DISMIC-25, 0.20-mm pore size;
ducted in an environment of 98% humidity at 37 C and Advantec, Tokyo, Japan) and the powder component
repeated 5 times. Mean value for each samples were via gamma irradiation. The components were then
computed to represent their respective setting time. mixed in a sterile environment. In this process, the sam-
ples were submerged in DMEM media and then seeded
Compressive strength. The samples were prepared to have with 1  105 cells/mL number of cells. A set of samples
a cylinder form (5 mm in diameter and 10 mm height). was incubated for 1, 3 and 5 days, respectively. MTT
The samples were placed in an incubator of 37 C and substrate (5 mg/mL) were supplied to cells and incu-
100% humidity for 7 days. After incubation, the sam- bated for 4 h. The MTT solution and media were
ples were measured for their compressive strengths then withdrawn and replaced with dimethyl sulfoxide
using a universal testing machine (R&B Unitech, to dissolve the formed formazan crystals. The plates
742 Journal of Biomaterials Applications 28(5)

were placed in a shaker for 1 h and the resulting solu- and washed twice by PBS after each incubation time.
tion was collected and transferred into 96-well plates, Using the Nucleospin RNA II Total RNA Isolation Kit
with each well containing 100 mL. Absorbance measure- (Germany), the total RNA from the collected cells were
ments were taken at 595 nm wavelengths through an then isolated. From the isolated RNA, cDNA was
ELISA reader (Infinite F50, Tecan). Cell viability was synthesized by using the iScriptTM cDNA synthesis
computed as the ratio of the number of cells in the Kit (Bio-Rad, USA). The resulting cDNA, 1 mL, was
treated wells to the number of cells in the untreated subjected to RT-PCR using iQ SYBR Green Supermix
wells prepared using fresh medium to serve as negative (Bio-Rad, USA). Another set of tests with the same
control. amount of cDNA was used to amplify the housekeep-
To visualize the cell growth, the fabricated samples ing gene, glyceraldehyde-3-phosphate dehydrogenase
were placed in 24-well culture plates. 1  105 cells were (GAPDH). RT-PCR subjected the samples to the fol-
then seeded onto the surfaces of the fabricated samples. lowing condition: pre-denaturation at 94 C for 3 min,
The media with the cells were then supplied to the well run through 40 cycles (denature 94 C/20 sec, annealing
until the samples were completely submerged (limited 60 C /20 sec, attention 72 C/30 sec) and then melting
to 1 mL). Different sets of samples were prepared by the analysis from 65 to 94 C at ramp rate of 0.5 C.
same procedure and were incubated up to 20 min,
60 min, 90 min, 1 day, 3 days and 5 days at 37 C,
95% humidity and 5% CO2. After each incubation
period, media was withdrawn and washed with PBS,
In vivo experiments
and then the samples were prepared for observation. Fabricated IBS samples were implanted to live animals
The samples were observed using SEM and confocal to assess their biocompatibility in vivo. Eight male New
microscope. Zealand white rabbits, averaging 3 kg, were used for
For SEM, the cell-seeded scaffolds were fixed and operations, 4 rabbits each for each sample of IBS-
dehydrated with different concentrations of ethanol BG0 and IBS-BG30. Before the operations, the animals
(50%, 60%, 70%, 80%, 90%, 95% and 100%). After were kept in a controlled environment and handled
dehydration, the samples were immersed in hexam- according to the procedures set by the
ethyldisialazane (HMDS, Sigma H4875) for critical Soonchunhyang Institutional Animal Care and Use
point drying and further air dried under the hood. Committee. Animals were individually kept in steel
The samples were mounted in a specimen holder and cages supplied with standard liquid and food for con-
coated with platinum. sumption. At the times of the operations, the rabbits
For confocal microscopy preparation, the samples were administered with anesthesia through intramuscu-
were fixed using 4% paraformaldehyde (Sigma, USA). lar injection. The femoral bone was drilled to produce a
The samples were then washed with PBS and permea- defect measuring 4 mm in diameter. The pre-sterilized
bilized with 0.5% Triton X-100. The nuclei of the cells samples were prepared and injected into the hole using
were stained using 40 -6-Diamidino-2-phenylindole a 3-mL syringe without a needle. The amount of sam-
(DAPI, Invitrogen) solution while the other parts of ples administered was limited to 0.2 mL. The cut was
the cells in the samples were stained using 3,30 -dihexy- re-sutured immediately and properly cleaned with povi-
loxacarbocyanine iodide (DiOC6, Invitrogen). The done iodine antiseptic. The rabbits were treated with
images taken were analyzed by using the FV10i-ASW antibiotics after the surgery to prevent infection. After
3.0 Viewer software. 3 months, the rabbits were sacrificed and the grafted
femoral bones were extracted. The harvested grafts
were covered with paraffin and each bone graft was
Molecular detection. Real-time polymerase chain reaction attached to the sample stage of SkyScan Desktop
(RT-PCR) was used to detect gene expression of primer Micro-CT 1172 (Aartselaar, Belgium). X-ray radio-
sequences for human protein such as osteocalcin (OC), graphs of the bone samples were taken with the
osteonectin (ON), collagen-1 (Col-1) and bone sialo- Micro-CT machine with a source voltage of 60 kV
protein (BSP). For this experiment, MG63 cells were and a current of 167 mA. The sample was rotated
grown in the surface of the samples containing 0% 360 , during which images were acquired every 0.4 .
and 30% bioactive glass material, respectively. The Images of cross-sectional slices of the samples were
samples were made to have measurement of 5 cm in taken. The resulting images were reconstructed at
diameter and height of 0.5 cm. Another set-up, in threshold values of 0.010–0.067 Hounsfield units in
which the cells were grown in plain DMEM media order to distinguish the bone from the hollow space.
only, was tested to serve as a positive control. The Micro-CT results were further evaluated using
cells grown in the surface of the IBS samples for 1, 7 SKyScan CTAn software, which analyzed the bone
and 14 days were then collected using trypsin EDTA parameters and properties.
Sadiasa et al. 743

The femurs of the rabbits were immersed in 5% (Figure 1(c)) shows peaks of Si and Na that can be
nitric acid solution for decalcification. After 48 h in attributed to the precursors used in the synthesis of
the solution, the parts of the bones containing the the bioactive glass material.
implanted samples were cut. The decalcified bones The IBS was fabricated successfully and varying
were dehydrated using a series of ethanol (70%, 80%, amounts of bioactive glass materials were incorporated
90% and 100%) and then washed with xylene to to the samples. The surfaces of the fabricated samples,
remove excess alcohol. The bone samples were as shown by SEM observations in Figure 2(b-1) and
embedded in paraffin wax then cut into 5 mm sections (b-2), were similar to that of typical cement, rough
using a microtome (HM 325, Thermoscientific). These and fractured. The surfaces of the samples showed irre-
sections were mounted in glass slides and were stained gular-size particles, including what seemed to be fine
with hematoxylin and eosin and Masson’s Trichrome powder to flakes-like structures. The particles observed
staining method. The stained slides were observed using in the IBS sample without bioactive glass have bigger
an optical microscope (Olympus BX51, Japan) and structures compared to the sample containing bioactive
their images were analyzed with the help of CellSens glass, which was covered by finer particles. The samples
software. were very compact but slightly showing presence of
varying sizes of pores. In the EDS profile taken using
SEM, trace amounts of Na and Si were observed in the
Statistical analyses profile of IBS-BG30, which indicated the presence of
Reported values of the different experiments were bioactive glass. Also, after the additions of bioactive
expressed as the mean average of 3 replicates with glass, the CA/P ratios of the samples increased with
standard deviation, unless otherwise stated. The results the peak of Ca more intensified.
were analyzed statistically using single factor and two-
way ANOVA with post-hoc correction (Tukey’s and
Bonferroni method). All analyses were carried out
Setting time
using GraphPad Prism 5 with a confidence level of The different components of the bone substitutes were
p  0.05 to determine the statistical significance of the mixed to form a paste-like mixture which has the ten-
data. dency to be molded. Setting time was measured from
the time a material was allowed to set to the time the
material hardened to presumably support stress. This
Results was the period of time from the end of mixing to the
Characterization of the bioactive glass and the time the material has set to a state in which a needle
fails to make a complete circular indentation in the
fabricated IBS cement.2
Figure 1(a) showed the FTIR spectra of the bioactive Figure 2(c) enumerates the setting time for each
glass exhibiting the abundance of glassy phase through sample. It can be observed that the addition of bio-
the presence of Si-O-Si vibration mode in the 1000– active glass material affected the setting time of an
1200 wave number regions. There is a brief vibration IBS. IBS-BG0 had the longest setting time of
corresponding to the non-bridging Si-O bond at around 43  2.5 min. The addition of bioactive glass material
900. Both the received powder (a-1) and the calcined decreased the setting times of IBS-BG10
powders at 600 C (a-2) showed the peak profiles with- (36  2.00 min), IBS-BG20 (28  2.00 min) and IBS-
out noticeable alteration. The peak at 1000 cm1 is BG30 (24.67  2.08 min).
assigned to the Si-O-Si asymmetric stretching mode,
whereas the vibration at 900 cm1 is associated with
symmetric Si-O-Si stretching or vibration modes of
Compressive strength
the silica ring structures.31
The bioactive glass was observed using SEM as Compressive strengths are shown in Figure 2(c).
shown in Figure 1(b). As shown in the image, the bio- Increasing concentration of bioactive glass material
active glass powder was seen to be composed of very resulted in increased compressive strength: IBS-BG0,
fine particles. Agglomeration of the powder particles without BG addition, recorded a compressive strength
was observed, thus making it hard for the definite meas- of 7.96  1.7 MPa; IBS-BG10 had a compressive
urement of the size of a single particle. If taking into strength of 10.83  1.9 MPa; IBS-BG20 13.80 
account the magnification of the image, the powder 1.12 MPa and IBS-BG30 15.04  2.4 MPa. The
particles appears very small and it can be generalized increases in the compressive strength due to the add-
that the fabricated bioactive glass powders are of ition of bioactive glass material was found to be signifi-
nanosize in measurement. EDS profile of the powder cant (p < 0.01; n ¼ 5).
744 Journal of Biomaterials Applications 28(5)

Figure 1. Fourier transform infrared spectroscopy (FTIR) spectra of the bioactive glass powders in (a-1) as received condition and
(a-2) after calcination and (b) scanning electron microscope (SEM) micrograph of the bioactive glass powder with an inset of a higher
magnification image and its (c) EDS profile.

Figure 2. Images of the injectable bone substitute (IBS): (a-1) injectability of the IBS (a-2) IBS samples after setting into cylindrical
shapes and scanning electron microscope (SEM) surfaces with respective EDS profile of (b-1) IBS-BG0 and (b-2) IBS-BG30. Setting time
and compressive strength of the IBS after 1 week of incubation depending on the concentration of bioactive glass. Mean  SD.
Setting time measurement: (p < 0.01; n ¼ 5); Compressive strength: (p < 0.01; n ¼ 5). * indicates statistically significant difference from
IBS-BG0.

Surface morphology and bioactivity experiments the case of SBF submersion show that the intensity of
the apatite formed varied with the percentage of bio-
using SBF
active glass material in the samples. More pronounced
Figure 3 shows the XRD profiles of the post set IBS peaks of HAp were observed especially in the pro-
after it had been incubated for 7 days. After incubation, longed submersion to SBF. Some residual peaks of
it was immersed in SBF solution. The XRD profiles for the TTCP and DCPD were observed, implying a
Sadiasa et al. 745

Figure 3. Post-setting x-ray diffraction (XRD) profiles of IBS-BG0 (a-0) before simulated body fluid (SBF) submersion, (a-1) after 1
day, (a-7) 7 days and (a-28) days of SBF submersion and IBS-BG30 (a-0) before SBF submersion, (a-1) after 1 day, (a-7) 7 days and (a-28)
days of SBF submersion.

possible incomplete conversion of the constituent mix- statistically (p < 0.05). However, it was observed that
ture to HAp. In the case of CSD, it tends to be missing there is no significant difference between cell prolifer-
in some profiles. Apatite formation after SBF forma- ation on IBS-BG20 and IBS-BG30.
tion was also confirmed using SEM. Figure 4 shows the As shown in Figure 8, SEM images were used to
surface morphologies of the samples that had been verify the ability of the fabricated samples to accom-
immersed to SBF and incubated in a 37 C incubator modate cellular attachment of MG63 cells and were
for 7 days. The images captured show crystal-like used to characterize the morphology of the attached
shapes on the surfaces of the samples, an evidence of cells. In the images, the cells are well attached to the
apatite formation. The formations were bigger and surfaces of the fabricated samples. Furthermore,
more vigorous in samples with higher concentrations cell density increases gradually on the surfaces of the
of bioactive glass material. The surfaces of all samples fabricated samples with the increase of bioactive glass
were generally rough and irregularly shaped. Big cracks material. Confocal images (Figure 9) show the nuclei of
and pores were present and the apatite formed with the cells stained with DAPI. These images were used to
crystal-like shape and pointed edges like needles. This demonstrate the proliferation behavior of the MG63
kind of surface morphology was also observed in our cells in the surfaces of the IBS samples: More cells
previous study which studied the effect of HPMC con- are attached to IBS-BG30 than the 3 other samples
centration on apatite formation.2 after 7 days of incubation.

Cell adhesion and proliferation Polymerase chain reaction

Figure 5 shows that the higher the bioactive glass The synthesized cDNA from the extracted RNA of the
material content in a sample, the more the cells prolif- MG63 cells grown in the surface of the samples was
erated in the surface of the samples. The effect of bio- used for detecting genes expression. The resulting
active glass material on cell adhesion (20, 60, 90 min) levels of gene expression were reported as fold change
and proliferation behavior (after 1–5 days) were (2
Ct) in relation to the expression levels recorded from
observed. Cell proliferation on the samples increased positive control or the calibrator. The result in Figure 7
as the concentration of bioactive glass increased. shows the cellular gene expression of the cells collected
Significant changes were observed in the proliferations from IBS-BG0 and IBS-BG30 samples. After 1 day of
of the cells grown in the surface of the samples contain- incubation, relatively insignificant difference was
ing bioactive glass in relation to IBS-BG0 as evaluated observed between the two samples across the
746 Journal of Biomaterials Applications 28(5)

Figure 4. Scanning electron microscope (SEM) images of IBSs’ surfaces after submersion in simulated body fluid (SBF) (a) IBS-BG0,
(b) IBS-BG10, (c) IBS-BG20 and (d) IBS-BG30 after 7 days of incubation. IBS: injectable bone substitutes.

Figure 5. Cell proliferation of MG63 cells in the samples after 1, 3 and 5 days of incubation: (a) as quantified by 3-[4, 5-
dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) showing significant effect of the bioactive glass concentration to cell
viability (Mean  SD, p < 0.05; n ¼ 5) and as shown by SEM images in the surface of IBS-BG0 after (b-1) 1 day, (b-2) 3 days and (b-3) 5
days of incubation and IBS-BG30 after (c-1) 1 day, (c-2) 3 days and (c-3) 5 days of incubation. IBS: injectable bone substitutes.
Sadiasa et al. 747

Figure 6. Confocal images of cell adhesion behavior and growth morphology of MG-63 cells on the surfaces of after (a) IBS-BG0, (b)
IBS-BG10, (c) IBS-BG20 and (d) IBS-BG30 after 7 days of incubation. Nuclei ¼ red color; cytoplasm ¼ green color. IBS: injectable bone
substitutes.

four primers. The cells from IBS-BG0 had higher 8(b-3 to 4)) showed a more porous and smaller material
expressions of OC (0.83  0.16) and BSP (1.05  0.18) representing the sample acquired from the femoral site
than the cells from IBS-BG30 (0.66  0.19 and implanted with IBS-BG30. Thicker cortical bone was
0.95  0.17, respectively). Further incubation of the also observed, covering the cavity in the sample site of
cells clarified of the effect of bioactive glass on gene IBS-BG30 than that of IBS-BG0. The differences in
expression. Significant differences in the amount of bone volume and tissue volume between the two sam-
gene expression were observed between IBS-BG0 and ples were also observed and were further analyzed
IBS-BG30, especially in the expression of BSP after 14 quantitatively (Table 1): the IBS-BG30 registered
days, wherein IBS-BG30 registered a value of higher percentage of bone volume, 78%, when calcu-
2.61  0.16, which is almost double of that of IBS- lated against the tissue volume than IBS-BG0, 61%.
BG0 (1.40  0.23). Histological staining of the sections using hematox-
ylin and eosin staining method (Figures 9 and 10)
showed that the sample sites were replaced with new
In vivo results bone tissue formations. As compared to the un-
After 3 months of implantation, the animals were implanted part, the newly-formed bone now covering
observed to be in healthy state. The rabbits were sacri- the sample site had the same morphology as human
ficed and the femoral bone of each animal containing bone, showing distinct characteristics such as osteons
the implant was extracted. X-ray tomographic images (Figures 9(a) and 10(a)). Spongy cell tissues were
of the sample sites clearly showed the integration of the attached to the sample, connecting to the cancellous
samples without bioactive glass (IBS-BG0) and with bone (Figures 9(b) and 10(b)). Several factors con-
bioactive glass (IBS-BG30) in the bones of the rabbits firmed bone formation, such as the formation of an
(Figure 8(a-1) and (b-1)). Micro-CT images of the sam- extracellular matrix and the presence of different bone
ples, IBS-BG0 and IBS-BG30 (Figure 8(a-2) and (b-2)), cells such as osteoblasts and osteoclasts type cells.
showed that smaller sample was seen in the sample site Figures 9(c) and 10(c) show the new bones that were
of the IBS-BG30 than that of IBS-BG0. Three-dimen- formed within the sample sites. The remains of the IBS
sional image reconstruction of Micro-CT scans of the samples were surrounded by native bone cells (Figure
samples and sample sites of IBS-BG0 and sample sites 9(d) and 10(d)). The remains of IBS-BG0 in the sample
of IBS-BG0 and IBS-BG30 (Figures 8(a-3 to 4) and site were bigger in size and number than those of the
748 Journal of Biomaterials Applications 28(5)

Figure 7. Real-time PCR analysis of osteogenic gene marker expression of MG63 cell line grown in the surface of IBS-BG0 and IBS-
BG30 after 1, 7 and 14 days: (a) Osteocalcin (p < 0.03) (b) Osteonectin (p < 0.03) (c) Collagen-1 (p < 0.04) and (d) bone sialoprotein.
(p < 0.04) Expression normalized to housekeeping gene GAPDH. Mean  SD; n ¼ 3. IBS: injectable bone substitutes.

Figure 8. Micro-CT image of bone implanted with (a) IBS-BG0 and (e) IBS-BG30; Micro-CT image of the sample site implanted with
(b) IBS-BG0 and (f) IBS-BG30 after 3 months; Constructed 3D representation of the sample site implanted with (c) IBS-BG0 and (g)
IBS-BG30 after 3 months; Constructed 3D representation of the (d) IBS-BG0 and (h) IBS-BG30 sample implanted in vivo after 3
months. IBS: injectable bone substitutes.
Sadiasa et al. 749

Table 1. Micro-CT 3D analysis of the injectable bone substitute Effects of the bioactive glass on the physical and
after 12 weeks.
mechanical properties of the IBS
IBS-BG0 IBS-BG30
It can be concluded that the bioactive glass was suc-
Tissue volume (mm3) 7.05  1.92 11.43  2.43 cessfully incorporated in the IBS system. In the EDS
Bone volume (mm3) 4.33  0.45 8.94  1.34 profiles presented in Figure 2(b-2), the presence of Na
Percent bone volume (%) 61.34  6.98 78.20  5.75 and Si in the IBS-BG30 profile signifies the incorpor-
ation of the bioactive glass to the IBS system as the
following elements were its primary components.
IBS-BG30 in the sample site. This may indicate better Also, after the addition of bioactive glass, the Ca/P
resorption rate of IBS-BG30 than IBS-BG0. Bone for- ratios of the samples increased with the peak of Ca
mation in the IBS-BG30 sample site was significantly more intensified. The addition of bioactive glass
different from that in the IBS-BG0 sample site; bone means more source of Ca while P remains the same.
tissues interacted well with the IBS-BG30 sample. No The effect of compositional variation of the bone sub-
inflammatory reaction and adherence of macrophages stitute on the mechanical behavior and biocompatibil-
were observed, indicating the biocompatibility of the ity of the bone substitute was evaluated.
IBS samples in vivo. Because IBS systems require short setting time after
Further analyses of the slides using Masson’s trich- mixing the components of the precursor materials, the
ome staining suggested the presence of collagen-rich setting times of the samples were determined.
regions in the sample sites of IBS-BG0 and IBS- Prolonged setting time may cause problems during clin-
BG30. Figures 11 and 12 show the sample sites of ical application when the material needs to show
IBS-BG0 and IBS-BG30, respectively. In these strength and integrity as soon as possible to support
images, traces of collagen were shown to be deposited stress and avoid disintegration.37 Usually, a light and
across the sample sites. Collagen deposits, represented thick needle is used to measure the initial setting time
by the blue stain in the slides, were found in the newly (I), while a heavier and thinner needle for the final set-
grown bone and even within the sample itself, ting time (F). In clinical aspect, it determines whether
surrounding the remains of the IBS. Figures 11(b) that the cement paste will be implanted before time I
and 12(b) show enlarged parts of the sample sites and that the wound can be closed after time F.38
where native bone cells and tissues interact with the A shorter final setting is required for a stable implant
remains of the IBS samples. New bones were formed after the operation. Too long final setting time may
(Figures 11(c) and 12(c)) within the sample sites, and impair the setting mechanism of the cement due to
native bone cells surrounded the remains of the IBS the invasion of the body fluid. On the other hand, meas-
(Figures 11(d) and 12(d)). Collagen appeared to be urement of the initial setting time will help evaluate the
more elaborate and distinct in the IBS-BG30 sample. onset of the setting action, which is necessary to deter-
Collagen was deposited, surrounding the remaining mine the handling time of the injectable paste/slurry.
samples and forming within the samples, which indi- However, we did not evaluate this in our system and
cated new bone formation. The collagen-rich regions the mixing of the components was all conducted for
further validated the biocompatibility of IBS contain- 1–2 min. In Figure 2(c4), the final setting time of each
ing bioactive glass in vivo. sample decreases as the concentration of bioactive glass
increases. In a previous study, the liquid component of
the fabricated samples, which is comprised of citric
Discussion acid, chitosan and HPMC, was found to directly
Bone healing by implant grafting is initiated by the affect the setting behavior of the IBS. The study rec-
colonization of osteogenic cells, which can promote the orded a setting time of around 35 min for their IBS
formation of new bone periphery. In order to facilitate system. Compared to this result, the IBS samples con-
faster healing, implanted material should be osteocon- taining bioactive glass recorded lower final setting
ductive, osteoinductive and osteogenic.32 Bioactive times, especially IBS-BG30 (24.67  2.08 min). The dif-
glass, with these properties, is considered to be a good ference between the samples of Thai et al. and IBS in
material for a bone substitute. However, bioactive glass this study was the citric acid concentration (Thai et.
has poor mechanical properties.33 Granular form of bio- al ¼ 20% citric acid; IBS-BG ¼ 10%).2 The citric acid
active glass can be difficult to handle and fill in cavities. contained in the liquid component can act as water-
Thus, it should be incorporated into a bone scaffold.34 In reducing agent, which was absorbed on the surfaces
this study, the newly developed bioactive glass system of the powder nuclei. This absorption was brought
was incorporated to the IBS from previous studies, about by the chelation reaction between the calcium
which improved the performance of the latter.2,35,36 ions (Ca2þ) from the powder component of the IBS
750 Journal of Biomaterials Applications 28(5)

Figure 9. Histological sections of implanted IBS-BG0 sample in a Rabbit’s femur stained using hematoxylin and eosin staining
method. (a) the sample site of IBS-BG0 after 3 months of implantation with image b, c and d as enlarged image of the area labeled P, Q
and R, respectively. It can be seen that the artificial defect made during implantation was now covered with a new bone as pointed by
the arrow; (b) the surface of interaction between the bone tissue and the sample as connected by fibroblast cells; (c) the new bone
formed within the sample site; (d) the remains of the IBS surrounded by native bone cells. BV: Blood vessel; NB: New Bone; IBS:
remains of the injectable bone substitute.

and the citric from the liquid component of the IBS. In (PO43). The particle size of the bioactive glass material
a scenario wherein the powder component absorbed the may also be critical in determining the compressive
citric acid molecules, the water molecules for hydration strength of the fabricated IBS samples. By increasing
were conserved and the surface area for dissolution was the concentration of the bioactive glass materials, the
increased.2,18 The resulting precipitation of the powder voids inside the bigger sized particles of the CPC was
component in the liquid component decreases the set- being filled up by the fine particles of the bioactive glass
ting time. The addition of the bioactive glass to the IBS powder, thus increasing the packing density and
increases the source of Ca2þ ions resulting to further decreasing the steric hindrance between particles.
decrease in the setting time of the material. The add- Because of this, the components of the IBS can be
ition of bioactive glass material particles in the IBS can packed easier and have a more compact composition.
also increase the surface tension and water retention In the previous study, the improve compressive strength
ability of the material because of two factors; the (1) of the IBS was attributed to the absorption mechanism
chemical changes introduced by the bioactive glass of the citric acid by the powder component, making the
material and the (2) reduction of pore volume that citric acid molecules a primary component of the
induces capillary actions which further affected the set- matrix.2 Due to the water-reducing effect of citric
ting mechanism.39 acid, the particles were arranged more tightly, which
The addition of bioactive glass was observed to also improved the particle packing and reduced the cement
improve the compressive strength of the IBS. The porosity.10 These effects of citric acid to the material
increase in the compressive strength of the material were further improved with the addition of bioactive
due to the addition of bioactive glass material can be glass material by further improving the water absorp-
brought upon by the increased resistance to dislocation tion capability of the powder due to the higher powder-
motion by lattice strains, which are established by local to-liquid ratio of the IBS containing bioactive glass
super saturation of Ca2þ ions as well as phosphate ions materials.
Sadiasa et al. 751

Figure 10. Histological sections of implanted IBS-BG30 sample in a Rabbit’s femur stained using hematoxylin and eosin staining
method. (a) the sample site of IBS-BG0 after 3 months of implantation with images b, c and d as enlarged image of the area labeled P, Q
and R, respectively. It can be seen that the artificial defect made during implantation was now covered with a new bone as pointed by
the arrow; (b) a new cortical bone formed surrounding the sample site; (c) new bone tissue starting to form in the middle of the
sample site surrounded by native bone cells; (d) the remains of the IBS surrounded by native bone cells. NB: New Bone; IBS: remains
of the injectable bone substitute.

In vitro biocompatibility of the IBS modified with

But with the expected acidity of citric acid, which is
bioactive glass detrimental to cell viability, bioactive glass was added
Several studies, especially those of Hench et al., have to the CPC powder to further elevate the biocompati-
described the reactions that occur in the surface of bio- bility of the bone substitute material. Figure 5 showed
active glasses.40–43 It was discussed that upon exposure increased cell proliferation with increasing bioactive
to bodily fluids, the bioactive glass will form a surface glass material concentration for every sampling day,
that is chemically and structurally similar to the natural which further validates the claim that bioactive glass
bone. This formation of apatite layer on the surfaces of improves the biocompatibility of the IBS, especially in
the samples is one of the prerequisite that a biomaterial comparison to the results of a previous study in our lab
has to satisfy if it is to bond to living tissue under by Jyoti et al., who tested an IBS system without bio-
physiological conditions.44 In this study, the SBF solu- active glass.35 As discussed, apatite formation increased
tion was prepared to mimic the environment inside a as the concentration of the bioactive glass in the sample
human body. The solution was prepared to have ionic increased. Based on this information, native bone cells
concentrations similar to those in human blood plasma, such as osteoblasts are expected to adhere and prolif-
which when used in vitro can reproduce in vivo surface erate in the fabricated IBS samples. In this experiment,
structure change.45 The result (Figures 5 and 6) showed MG63 cells, osteoblast-like cells, were seeded on the
more apparent apatite formation as the concentration sample surface. The cells were attached to the surface
of bioactive glass increases. Some peaks of CSD either of IBS-BG30 by several actin-like filaments and cyto-
decreased in intensity or eliminated as there is the pos- plasmic extension. Cell adhesion and proliferation were
sibility of CSD to dissolve in SBF solution as it exhibits seemingly promoted with increasing percentage of bio-
high dissolution rate. active glass in the sample. This observation was further
In general, the conventional CPC and the products supported by the images of cell proliferation in the sam-
produced in its reaction do not elicit cytotoxic effects. ples for longer incubation times. Cell material
752 Journal of Biomaterials Applications 28(5)

Figure 11. Histological sections of implanted IBS-BG0 sample in a Rabbit’s femur stained using Masson’s trichome staining method.
The images show the collagen deposited across the sample site indicated by the blue stains. (a) the sample site of IBS-BG0 after 3
months of implantation with images b and c as enlarged image of the area labeled P and Q, respectively; (b) the surface of interaction
between the bone tissue and the sample with image (d) as enlarged image of the area labeled R; (c) new bone tissue forming within the
sample site surrounded by native bone cells; (d) the remains of the IBS surrounded by native bone cells. Remains of the IBS stained as
red to violet in the Figures 11b and 11d with the collagen surrounding or within it indicating good interaction of the sample in vivo. NB:
New Bone; IBS: remains of the injectable bone substitute.

deposition increased as the concentration of bioactive gene of interest.46 Ct values of the GAPDH in three
glass material increased for different incubation times. samples were almost the same and their melting
The cells in IBS-BG30 proliferated more vigorously points indicated correct gene expression of the target
than those in IBS-BG0. Small, rounded cells were sequence. Osteoblasts produce most of the proteins in a
also noted, suggesting the production of younger cells bone extracellular matrix, including Col-1 and BSP,
and continuous proliferation. Bigger cells were and control their mineralization. Osteocalcin is a 5-
observed after longer incubation, and the confocal kDa, hydroxyapatite-binding non-collagenous protein
images of DAPI-stained samples defined the nuclei of of osteoblasts. It is synthesized in osteoblasts and oden-
the cells attached to the sample surfaces. As a result, the toblasts and is the most gene-specific of all osteoblasts.
number of cells per field could be better estimated: the In some in vitro studies, it was shown that ON serves a
IBS-BG30 had significantly more cells than IBS-BG0. role in the modulation of cell division and cell migra-
To further investigate the relative biocompatibility tion and in the initiation of active mineralization in
of the fabricated materials, the IBS systems were sub- normal skeletal tissue.47–49 Due to this factor, osteo-
jected to RT-PCR. The possible effect of the sample on blasts are considered to be highly suitable for demon-
the expression of bone cell-specific markers, namely strating cell-material interactions in bone substitutes as
OC, ON, Col-1 and BSP, were assessed and observed. a phenotypic marker. The expressions of gene primers
GAPDH was used as a housekeeping gene to normalize were successful and the levels of expression of OC, ON,
the target gene expression data as this type of gene is Col-1 and BSP were higher in IBS-BG30 than in IBS-
usually stable from treatment to treatment. With the BG0. Therefore, IBS-BG30 contained higher amount
use of GAPDH, it can be observed whether there was of starting DNA than IBS-BG0, signifying that the
a significant variation in the expression level in a cells proliferated better in the set-up with bioactive
particular set of samples due to a difference in the glass than that without bioactive glass.
Sadiasa et al. 753

Figure 12. Histological sections of implanted IBS-BG0 sample in a Rabbit’s femur stained using Masson’s trichome staining method.
The images show the collagen deposited across the sample site indicated by the blue stains. (a) the sample site of IBS-BG30 after 3
months of implantation with images b, c and d as enlarged image of the area labeled P, Q and R, respectively; (b) the surface of
interaction between a newly formed cortical bone surrounding the sample site and the sample (c) new bone tissue formed within the
sample site surrounded by native bone cells; (d) the remains of the IBS and native bone tissues starting to form. It can be observed that
collagen deposit can be found around and within the sample site. Relatively heavier density of collagen can be observed in the sample
indicating better bone growth. NB: new bone; IBS: remains of the injectable bone substitute.

In vivo analysis of the IBS modified with bioactive samples were osteoconductive and could promote the
proliferation of osteogenic cells. In addition, the deg-
glass
radation of the samples was observed, wherein the IBS-
In vivo examination of the samples implanted in the BG30 became more porous and smaller, suggesting that
rabbit’s bone supported the idea of better healing pro- the IBS-containing bioactive glass was more biodegrad-
cess and bone growth with increased bioactive glass able than the IBS not containing bioactive glass. This
content. The implantation time was decided to be 3 observation is supported by the constructed 3D repre-
months. The IBS is intended to fill the void and sentation of the IBS-BG0 sample (Figure 8(a-4)) and
defect site without creating porous space. Therefore, IBS-BG30 sample (Figure 8(b-4)) in which a smaller
the resorption of the implant and subsequent new and more porous IBS remains were observed in the
bone formations replacing it need to be investigated sample site of IBS-BG30. The pronounced loss of
also. It was thought of that 2 months will be the right mass sample in the site of the IBS-BG30 compared to
implantation time to investigate bone growth in a the sample site of IBS-BG0 suggested better resorbabil-
porous scaffold. But the IBS system is supposed to be ity and degradation of the IBS with bioactive glass.
compact and solid after setting, thus a longer implant- This supported the observation of Renno et al. wherein
ation time will reveal more about the degradation bioactive glass increases the degradation rate of CPC
behavior of the IBS samples and the bone growth it and CPC/PLGA composite cements. The degradation
promoted. Higher percentage of bone volume was rate of the material was associated with the dissolution
observed in the implanted sample site of IBS-BG30. rate of bioactive glass upon immediate contact with
This means more bones were produced, which is indi- fluids that have been observed to be faster than the
cation of better bone growth. This suggested that the slow-degradable pure CPC.50 The theory that bioactive
754 Journal of Biomaterials Applications 28(5)

glass promotes better bone healing was further con- Acknowledgement

firmed by the histological staining of the sample site. This research was supported by Soonchunhyang University
New bone formations were observed covering the Research Fund (NO20101214). The authors would also like
implanted site, indicating bone healing and regener- to acknowledge the contribution of Mr. Kim Shin Woo in the
ation. This healing and regeneration was also proven in vivo experiment.
by the collagen deposits found in the new bones formed
around and within the area of the sample sites. Native
bone cells were observed to be present and can be seen References
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