Fabrication of in-situ foamed chitosan/b-TCP scaffolds for bone tissue

engineering application
Martyna Kucharska
a,n
, Beata Butruk
a
, Katarzyna Walenko
a
, Tomasz Brynk
b
, Tomasz Ciach
a
a
Biomedical Engineering Laboratory, Faculty of Chemical and Process Engineering, Warsaw University of Technology, Warynskiego 1, 00-645 Warszawa, Poland
b
Faculty of Materials Science and Engineering, Warsaw University of Technology, Wo!oska 141, 02-507 Warszawa, Poland
a r t i c l e i n f o
Article history:
Received 6 March 2012
Accepted 1 July 2012
Available online 7 July 2012
Keywords:
Bone tissue engineering
Foaming
Chitosan
Tricalcium phosphate
a b s t r a c t
The present work describes the manufacturing process of biodegradable porous material for bone tissue
engineering application. The matrixes were produced by chemical foaming of chitosan solutions (CH).
The method was based on CO
2
release directly in the polymer by means of reaction between acetic acid
and sodium bicarbonate acting as a foaming agent. The method allowed obtaining the materials with
pores ranging from 10 to 1000 mm and inner porosity close to 90%. The degradability of the materials
was inuenced by TCP content and decreased with extending inorganic phase contribution. Along with
calcium phosphate content the Young modulus values were enclosed in the range of 0.020.5 MPa.
Preliminary cytocompatibilty studies revealed cell afnity of the materials manufactured.
& 2012 Elsevier B.V. All rights reserved.
1. Introduction
Tissue engineering is an interdisciplinary research eld, which
adapts the principles of engineering and biological sciences to
manufacture-engineered structures that can restore, maintain or
improve the tissues function [1,2]. Chitosan is the co-polymer of
D-glucosamine and N-acetyl D-glucosamine. It is biodegradable,
non-toxic and it possesses antibacterial properties. A lot of
research concerning the potential use of chitosan in medicine
has been published [39]. Due to its exceptional properties it was
appreciated and successfully employed as a drug carrier in the
forms of tablets, microspheres and nanoparticles [3,4,8]. A lot of
attention has been paid to the employment of chitosan in bone
tissue engineering scaffolds fabrication. In order to enhance
bioactivity of the scaffolds different inorganic llers have been
applied. Most commonly calcium phosphates such as hydroxya-
patite and b-tricalcium phosphates have been investigated and
formed into 3D composite matrixes [1012]. Various methods of
producing porous chitosan scaffold have been proposed so far.
There were papers describing the application of freeze-drying
method [13], thorough thermally induced phase separation [14],
up to more sophisticated techniques like agglomeration of chit-
osan microspheres [15,16]. One of the most popular approaches
for making porous chitosan scaffolds relates to freeze-drying
methodology of the polymer solution. The technique allows
obtaining materials with small pore sizes ranging from 20 to
100 mm [13,17], what might be insufcient for cell migration and
new bone tissue ingrowth [18,19].
The aim of the study was to propose a simple method for
fabrication of porous chitosan structures for bone tissue engineer-
ing application. A simple reaction between the acid present in the
chitosan solution and sodium bicarbonate, added as a foaming
agent, was applied in order to foam the polymer. The authors
produced, characterized and described the materials enriched
with osteoconductive b-TCP and discussed the inuence of
ceramics content on physical and biological properties of the
foams fabricated following the presented method.
2. Materials and methods
Fabrication process: The matrixes were derived from a 5% w/v
chitosan solution (Fluka, medium viscous; 4400 MPa s) prepared
in a 4% v/v acetic acid (Chempur, Poland). The amounts were
chosen experimentally. The dissolved chitosan was placed
in 100 mL plastic containers. Then, the b-TCP powder form
(35 mm particle size, Fluka) was added directly to the solution
and blended, so as to ensure even distribution. b-TCP was added
in quantities of 15%, 20%, 30%, 40% and 50% w/v and nal samples
were marked as CH, CH/15TCP, CH/20TCP, CH/40TCP and
CH/50TCP, respectively. After mixing all of the components,
sodium bicarbonate (NaHCO
3
, Poch, Poland) was addedacting
as a foaming agent according to the following reaction
CH
3
COOHNaHCO
3
-CH
3
COONaCO
2
H
2
O
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/matlet
Materials Letters
0167-577X/$ - see front matter & 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.matlet.2012.07.002
n
Corresponding author. Tel.: 48 22 234 62 79.
E-mail addresses: m.kucharska@ichip.pw.edu.pl,
martyna.kucharska@gmail.com (M. Kucharska).
Materials Letters 85 (2012) 124127
The bicarbonate was added stoichiometrically. Final solutions
were carefully blended, so as to ensure precise distribution of the
ingredients, and then freeze-dried. Finally, the dried samples
were being washed with distilled water for 24 h. During that
process, the medium was changed a few times, so that all residues
of components were washed away.
Physical and biological characterization: The sponges obtained in
the way described above were investigated, so that their physical,
chemical and biological properties could be characterized. The
assessment of morphology was conducted using microscopic
techniquesthe optical microscope Nikon Eclipse 80i and SEM
Hitachi TM-1000. The porosity of the materials was determined by
hydrostatic weighing (Mettler Toledo Excellence Plus XP) based on
the Archimedes principle. The interconnectivity of the pores was
evaluated with the use of microtomography X-ray scanning
(mCT, SkyScan 1174). It should be mentioned that materials made
solely from chitosan were fragile and difcult to form replicable
shapes, which made them difcult to study. Therefore, only
the results concerning their morphology are discussed herein. The
degradation of materials was examined in the conditions simulat-
ing physiological environment (PBS buffer, lysozyme 0.5 mg/ml,
temperature T37 1C) and expressed as the remaining dry weight
of the materials. The samples were subjected to compression tests
using universal electromechanical testing stand MTS Q/ test 10.
Before the test, the study materials were cut into same dimensions:
1.5 cm1.0 cm1.0 cm. On the basis of stressstrain characteristics
the Young modulus and compression strength were established. In
order to evaluate the cytotoxicity and the effect of the fabricated
materials on cells morphology, surface attachment and their viabi-
lity, an in vitro study using MG-63 cell line was conducted.
The results were described as optical density measured by XTT assay
and performed as the viability of the cells. Materials were sterilized
by electron radiation exposure before incubation. Cells seeded on the
scaffolds were incubated at 37 1C in 5% CO
2
. During the culture, the
cells morphology was monitored with the use of an optical micro-
scope. On the 3rd day of culture, the XTT test was conducted with
the incubation time of about 4 h. Then, the absorbance at 450 nm
wavelength was measured. The cells were preserved with glutar-
aldehyde and treated by Hoechst, so that the cells distribution on
material surface could be observed.
3. Results
Morphology of the materials, inuence of TCP content: The
conducted research revealed that the porosity of fabricated
Fig. 1. Comparison of material morphology and pore architecture with increasing concentration of b-TCP: (a) CH, (b) CH/15TCP, (c) CH/20TCP, (d) CH/40TCP, and (e), CH/50TCP.
M. Kucharska et al. / Materials Letters 85 (2012) 124127 125
sponges is close to 90%. The highly porous materials were obtained
by two methods: CO
2
release and the freeze-drying method. No
signicant relation between material volumetric porosity, marked
by Archimedes principle, and TCP content was observed. Never-
theless, it was discovered that ceramics (b-TCP) concentration
visibly affected the formation and size of the pores, which can be
conrmed by Fig. 1. At its lower concentration, the pores were more
irregular and the formation of macropores, with a diameter length
up to several millimeters, were dominant. In contrast, along with
increasing TCP content the pore architecture was more structured
and their dimensions were more regular (Fig. 1). It is also worth
stressing that TCP concentration inuencing pore formation at the
same time affected the macrostructure of the foams. The pore sizes
were measured by considering SEM and is presented in Fig. 2, they
range from 10 to 1000 mm. The presence of macropores was mostly
observed at lower TCP density. As presented in the pictures, the
materials containing higher concentration of ceramics are generally
better formed, compact, and have a dened geometrical shape and
regular pore size with homogenous distribution. Interconnectivity of
the pores and their open character were also evaluated by means of
mCT studies Fig. 2c.
Degradation: The degradation of the fabricated materials
proceeded as shown in Fig. 3. The largest weight loss was
observed on the 20th day of experiments for the samples with
the lowest content of ceramicsthe remaining dry weight
amounted to 40%. Materials with the highest concentration of
TCP revealed remaining dry mass residue of about 80%.
Compression test: Mechanical tests showed that ceramics
content signicantly affected the materials stiffness. Along with
the increasing amount of calcium phosphate materials tend to be
less elastic, which can be supported by Fig. 4. The materials
Fig. 3. Degradation of the materials containing different concentrations of TCP.
Fig. 2. SEM microphotographs of CH/50TCP material, architecture and size of the pores (a), b-TCP particles distribution in the material matrix (b), porous architecture
observed by mCT, shown in one of the cross-sections obtained (c).
Fig. 4. Inuence of TCP content on mechanical properties of fabricated sponges.
M. Kucharska et al. / Materials Letters 85 (2012) 124127 126
containing 50% of TCP revealed the Young modulus values at
about 0.5 MPa, while those with 15% of TCPonly about
0.02 MPa. Ceramics concentration also had a visible inuence
on compression strength of the spongesthe higher the concen-
tration of TCP, the higher the compression strength of the foams
was conrmed. Most probably the relation obtained was coming
from the reduction of pore sizes which decreased with increasing
TCP concentration.
Cytotoxicity: The viability of MG63 cell line cultured within the
materials after 3 days of incubation is shown in Fig. 5b. The XTT
studies showed that the highest living cell number was observed
in the sample CH/15TCP containing the lowest concentration of
TCP. The viability for the rest of the materials (Fig. 5b) was
denitely lower and hardly reached 20% of the control sample.
This might be explained by increasing roughness of the materials
coming from TCP content, what inuenced initial cells attach-
ment. Nevertheless, the cells cultured within the scaffolds were
monitored with respect to their morphology during the incuba-
tion and they were conrmed as well attened with correctly
developed osteoblastic phenotype. The distribution of the cells is
shown in Fig. 5a.
4. Conclusions
Novel method of porous materials manufacturing, based on in-
situ foaming is presented herein. The technique provides a
possibility to generate highly porous matrixes with porosity close
to 90% and pores ranging from 10 up to 1000 mm, creating
suitable microenvironment for cell transport and new bone tissue
ingrowth, when compared to the typical freeze-drying technique.
The morphology, degradation and mechanical properties of the
fabricated sponges were strongly affected by TCP concentration.
The materials obtained by the methodology presented revealed
cell afnity.
Acknowledgment
This work has been supported by the European Union in the
framework of European Social Fund through the Warsaw Uni-
versity of Technology Development Program.
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Fig. 5. Distribution of MG-63 cells Hoechst stained on the material containing 15% of TCP (a). Viability of MG63 cells after 3 days of culture on chitosan reinforced with
different TCP concentrations (b).
M. Kucharska et al. / Materials Letters 85 (2012) 124127 127