Journal of Biomaterials and Nanobiotechnology, 2013, 4, 316-326
http://dx.doi.org/10.4236/jbnb.2013.44040 Published Online October 2013 (http://www.scirp.org/journal/jbnb)
A Novel in Vitro Three-Dimensional Macroporous
Scaffolds from Bacterial Cellulose for Culture of
Breast Cancer Cells
Guangyao Xiong1, Honglin Luo2, Feng Gu3,4,5, Jing Zhang2, Da Hu2, Yizao Wan2*
1School of Mechanical and Electrical Engineering, East China Jiaotong University, Nanchang, China; 2Tianjin Key Laboratory of
Composite and Functional Materials, School of Materials Science and Engineering, Tianjin University, Tianjin, China; 3Department
of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China; 4Key
Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin, China; 5Key
Laboratory of Cancer Prevention and Therapy, Tianjin, China.
Received June 15th, 2013; revised July 15th, 2013; accepted August 2nd, 2013
Copyright © 2013 Guangyao Xiong et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
In this work, patterned macropores with a diameter larger than 100 μm were introduced to pristine three-dimensional
(3D) nanofibrous bacterial cellulose (BC) scaffolds by using the infrared laser micromachining technique in an attempt
to create an in vitro model for the culture of breast cancer cells. The morphology, pore structure, and mechanical per-
formance of the obtained patterned macroporous BC (PM-BC) scaffolds were characterized by scanning electron mi-
croscopy (SEM), mercury intrusion porosimeter, and mechanical testing. A human breast cancer cell (MDA-MB-231)
line was cultured onto the PM-BC scaffolds to investigate the role of macropores in the control of cancer cell behavior.
MTT assay, SEM, and hematoxylin and eosin (H&E) staining were employed to determine cell adhesion, growth, pro-
liferation, and infiltration. The PM-BC scaffolds were found to be able to promote cellular adhesion and proliferation on
the scaffolds, and further to allow for cell infiltration into the PM-BC scaffolds. The results demonstrated that BC scaf-
folds with laser-patterned macropores were promising for the in vitro 3D culture of breast cancer cells.
Keywords: 3D Culture; Scaffold; Bacterial Cellulose; Cancer Cell; Macropore
1. Introduction
Tissue engineering, as stated by Langer and Vacanti, is
the process of creating functional three-dimensional (3D)
tissues using scaffolds or devices that facilitate cell
growth, organization, and differentiation [1]. Tumor en-
gineering was described by Ghajar and Bissell as “the
construction of complex cell culture models that reca-
pitulate aspects of the in vivo tumor microenvironment to
study the dynamics of tumor development, progression,
and therapy on multiple scales” [2]. In tissue engineering,
a scaffold with in vivo architecture and in vivo microen-
vironments should be employed in order to create a bio-
logical tissue or organ with natural functions. In this
context, 3D scaffolds have been widely employed from a
biomimetics point of view. The biomimetic strategies
have also been employed in tumor engineering in the past
few years and thus 3D scaffolds have begun their appli-
cations in cancer research [3]. Use of 3D scaffolds in
tumor engineering has helped researchers realize that
there are significant differences between conventional
two-dimensional (2D) petri dish cultures and 3D cultures
and the latter can provide an ideal tool for the study of
the transformation of normal cells into cancer and tu-
morigenesis under pathologically relevant culture condi-
tions [4,5]. Furthermore, Bissell et al. commented that
appropriate 3D cultures could provide a more physio-
logically relevant approach to the analysis of gene func-
tion and cell phenotype ex vivo [4] while cancer cells
cultured in 2D poorly represented their in vivo physio-
logical conditions. For instance, the ability of malignant
cells to grow and metastasize in vivo depends upon spe-
cific cell-cell and cell-extracellular matrix (ECM) inter-
actions, many of which are absent when cells are cul-
tured on conventional 2D tissue culture plastic [6]. Addi-
tionally, it is well documented that the efficacy of poten-
tial anticancer drugs during preclinical development is
*Corresponding author.

A Novel in Vitro Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of
Breast Cancer Cells
317
generally tested in vitro using cancer cells grown in scaf-
folds. Conventional evaluation is based upon the 2D cul-
ture; however, a significant discrepancy in their efficacy
is observed when these drugs are evaluated in vivo. For
instance, Horning et al. declared that the cells grown in
3D scaffolds are more resistant to chemotherapy than
those grown in 2D culture [7]. Therefore, creating 3D
culture which mimics in vivo conditions is of primary
importance to the evaluation of the efficacy of anticancer
drugs as well as the study of tumor biology [8].
To date, various biomaterials including natural and
synthetic polymers have been used to fabricate 3D scaf-
folds for cancer research and anticancer drugs tests. Col-
lagen has been widely utilized to culture cells owing to
its excellent characteristics, including biocompatibility,
mechanical strength, degradability and limited immuno-
genicity [9,10]. Other biomaterials such as chitosanalgi-
nate [11], polyacrylamide [12], poly (lactic acid) (PLA)
[7,13], poly(lactic-co-glycolide) (PLGA) [7,13], hyalu-
ronan [14], and silk fibroin protein [15] have been ex-
plored as 3D models for cancer research.
Over the last two decades, various techniques have
been developed to fabricate biomimetic scaffolds for tis-
sue engineering and later for tumor engineering. Elec-
trospinning has been the most widely used technique to
create 3D scaffolds composed of nanofibers or mostly
sub-microfibers [16,17]. Electrospinning is a relatively
simple and scalable nanotechnological method for the
generation of nanostructured scaffolds that closely mimic
the dimensions of collagen fibrils of ECM and a very
recent report showed the capability of creating 3D scaf-
folds [18]. However, electrospun fibers do not allow pro-
per infiltration of the cells to the core of the scaffolds due
to the limited pore size. Another substantial disadvantage
is that the diameters of the fibers are usually at the upper
limits of the 50 - 500 nm range seen in natural ECM [19].
Therefore, scaffolds which allow effective cell seeding
and penetration, have controllable fiber diameter in nano-
scale and sufficient material properties are highly desir-
able in both tissue engineering and tumor engineering.
These requirements and the tremendous interest in ex-
ploring the potency of biomimetic scaffolds have urged
researchers to continuously develop more techniques.
Molecular self assembly [20] and phase separation [21,
22] have emerged as other promising techniques for the
fabrication of 3D nanofibrous scaffolds. Efforts are still
being made to find more alternative methodologies so as
to simultaneously control morphological, mechanical, and
chemical performances of scaffolds.
It is noteworthy that bacterial cellulose (BC), a natural
nanofibrous polymer, has attracted more and more atten-
tion. BC is not fabricated by electrospinning; instead it is
synthesized extracellularly by the bacterium Acetobacter
xylinum. BC has received enormous research interest and
the number of reports on BC has showed a tremendous
increase over recent years (reviewed by Petersen and
Gatenholm [23], and by Klemm and colleagues [24]). BC
nanofibers can have a diameter as small as 10 nm which
is the low limit of natural ECM fibers ranging from 10 to
several hundreds of nanometers [19]. BC is superior to
plant cellulose (PC) owing to its high purity, 3D mor-
phology, high crystallinity, high tensile strength and
modulus (the effective modulus of single fibrils of BC
ranged from 79 to 88 GPa versus 29 to 36 GPa for plant
cellulose [25]) in combination with a variety of proper-
ties such as high water holding capacity, large surface
area, and particularly good biocompatibility. For instance,
a long-term biocompatibility study conducted by Pertile
et al. confirmed that BC caused a mild and benign in-
flammatory reaction that decreased along time and did
not elicit a foreign body reaction [26]. Very recently, a
long-term study on in vivo biocompatibility of BC has
been reported and BC was defined as a biocompatible
material [27]. A latest study by Favi et al. showed that
BC scaffolds were cytocompatible and could support
cellular adhesion and proliferation, and allowed for os-
teogenic and chondrogenic differentiation of equine-
derived bone marrow mesenchymal stem cells (EqMSCs)
[28]. Shi and co-workers declared that BC was a good
localized delivery system for bone morphogenetic pro-
tein-2 (BMP) and would be a potential candidate in bone
tissue engineering [29]. Another study by Saska et al.
found that BC membranes functionalized with osteogenic
growth peptide (OGP) and its C-terminal pentapeptide
OGP could be used in bone tissue engineering/regenera-
tion [30]. Very recently, BC microstrands were fabricated
which could serve as a pathway of nutrition and oxygen
to feed the cells in the central region of a macroscopic
tissue [31]. Though extensive research has been carried
out to determine the potential of BC as tissue engineering
scaffolds, investigation on BC scaffolds for tumor engi-
neering has been very limited. The only pioneering study
on the in vitro culture of cancer cells including the hu-
man androgen-independent prostate cancer cell line (PC-
3), murine renal cancer cell line (RENCA), and human
breast cancer cell line (MDA-MB-231) on BC scaffolds
demonstrated that these cancer cells cultured on BC did
not have observable protrusions indicating undesirable
cancer cell responses, which were ascribed to the absence
of manufactured large porosity [32].
It has been accepted that pore structure is an essential
consideration in the development of scaffolds for tissue
engineering and pores must be interconnected and large
enough to allow for cell growth, migration and nutrient
flow, and for vascularization, new tissue formation and
remodeling so as to facilitate host tissue integration upon

A Novel in Vitro Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of
Breast Cancer Cells
318
2.2. Preparation of BC Pellicles implantation. Furthermore, pore volume (porosity), shape
and distribution also should be considered [33]. A recent
study demonstrated that the acrylate copolymers-based
scaffolds with aligned channels achieved a uniform colo-
nization by neural cells [34]. Our previous work indi-
cated that the BC/Gelatin/Hydroxyapatite scaffolds with
patterned pores supported the attachment and prolifera-
tion of chondrogenic rat cell [35]. However, how the
aligned pore channels affect cancer cell behavior has not
been reported.
The preparation and purification procedures of BC pelli-
cles were described previously [37,38]. Briefly, the bac-
terial strain, Acetobacter xylinum X-2, was grown in the
culture media containing 0.3 wt% green tea powder
(analytical grade) and 5 wt% sucrose (analytical grade)
for 7 days. The pH of the medium was adjusted to 4.5 by
acetic acid. BC pellicles were purified by soaking in de-
ionized water at 90˚C for 2 h followed by boiling in a 0.5
M NaOH solution for 15 min. The BC pellicles with 9
cm in diameter were then washed with deionized water
several times and soaked in 1% NaOH for 2 days. Finally,
the BC pellicles were washed free of alkali.
Therefore, the purpose of the present study was, for
the first time, to determine the feasibility of the BC scaf-
folds with aligned channels as effective in vitro cancer
models. To this end, the BC scaffolds with patterned ma-
cropores (with a diameter greater than 100 μm according
to literatures [33,36]) were fabricated by an infrared laser
micromachining technique and a human breast cancer
cell line (MDA-MB-231), as a model cancer cell line,
was seeded onto the 3D macroporous BC scaffolds to
investigate the cancer cellular responses to the scaffolds.
The cell behavior on these 3D macroporous BC scaffolds
was compared with that on the pristine BC scaffolds
[32].
2.3. Preparation of 3D BC Scaffolds with
Patterned Macropores via Laser Technology
Many previous studies reported the creation of patterned
pores in solid materials such as titanium [39,40], and
biodegradable polymers [41-43] mostly by using ultra-
violet (UV) or femtosecond laser micromachining. In this
study, patterned pores in BC hydrogels were created by
using infrared laser. Figure 1 illustrates the laser-micro-
machining process. The predetermined patterns (includ-
ing distance between neighboring pores and pore diame-
ter) were initially designed by a commercial CAD soft-
ware and input to a computer. The BC hydrogels were
then perforated using a CO2 excimer laser (wavelength
10.6 μm) according to the designed patterns to obtain BC
scaffolds with patterned macropores (named as PM-BC
hereinafter). The laser generated a stable power of 80 W,
the exposure time was about 1 - 2 s for every pore de-
pending on the thickness of the BC samples and the pore
diameter could be altered by adjusting the distance be-
tween specimens and laser focus (i.e. parameter d in
Figure 1). In this work, three PM-BC scaffolds with dif-
ferent pore size and pore density were fabricated and
2. Materials and Methods
2.1. Materials
The reagents used in this work included glucose, peptone,
yeast extract, disdium phosphate and acetate acid (Acros,
Biochemical), deionized water (Aqoapro CO., Ltd, Chong-
qing, China), sodium hydroxide, ethyl alcohol, formal-
dehyde, glutaraldehyde, sodium chloride (Tianjin Tianda
Tianlong Sci. & Tech. Co., Ltd., Tianjin, China), hank’s
balanced salt solution (HBSS), phosphate buffered saline
(PBS), dimethyl sulfoxide (DMSO, Gibco), and gelatin
(Sigma, analytical grade). All chemicals were used as
received without further purification.
Figure 1. Schematic setup of the laser-patterning system used in this work (the parameter d refers to the distance from laser
focus to the specimen).

A Novel in Vitro Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of
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319
their designations and parameters are listed in Table 1.
The resultant PM-BC scaffolds, as well as the pristine
BC scaffolds without macropores, were immersed into
deionized water for 2 h and then washed with distilled
water for several times.
2.4. Scaffold Characterization
2.4.1. Field Emission Scanning Electron
Microscopy (F E -SEM )
The morphology of the PM-BC scaffolds was observed
by using a Nano 430 field emission scanning electron
microscope (FE-SEM), FEI, USA. For FE-SEM observa-
tions, samples were sputter coated with gold and were
observed at an accelerating voltage of 10 kV.
2.4.2. Mercury Porosimeter
The pore size distribution and porosity of the BC scaf-
folds were determined by a PoreMaster 60 GT mercury
intrusion porosimeter (Quantachrome) that could meas-
ure pore diameter ranging from 950 μm to up to 3.6 nm.
2.4.3. Mechanical Te sting
The tensile properties of PM-BC and pristine BC sam-
ples in the wet state were determined using a Testometric
universal testing machine M350 (Testometric Co. Ltd.,
United Kingdom) in accordance with ASTM D 638-98
under ambient temperature and humidity (20˚C/65% RH)
with a constant crosshead speed of 5 mm/min. The ten-
sile modulus was determined from the linear region of
stress-strain curves. The Young’s modulus, tensile strength,
and strain at break were determined from at least five
samples.
2.5. Cell Studies
2.5.1. MTT Proliferation Assay
The cell proliferation was evaluated by the colorimetric
MTT assay. Firstly, the human breast cancer cell line
(MDA-MB-231) was maintained in DMEM (Gibco) with
10% FBS (Gibco) at 37˚C in a 5% CO2 incubator.
Monolayer MDA-MB-231 cells were harvested by tryp-
sin/EDTA treatment. Before cell seeding, cylindrical BC
scaffolds (Ф10 × 1 mm) were sterilized with UV radia-
tion. After sterilization, the scaffolds were pre-soaked in
Table 1. The designation of various samples prepared in
this work.
Sample No. Designed pore diameter
(μm)
Designed pore distance
(mm)
PM-BC-1 150 1.0
PM-BC-2 150 1.5
PM-BC-3 300 1.5
DMEM for at least 12 h. Subsequently, the scaffolds
were incubated in 24-well tissue culture plates with
MDA-MB-231 cells at a density of 2 × 105 cell/mL for 1,
3, 5 and 7 days at 37˚C in 5% CO2 incubator. After in-
cubation, the cell-scaffold constructs were rinsed with
PBS to remove non-adhering cells, followed by incuba-
tion in 50 μL MTT reagent for 4 h under the same condi-
tions as described above. After removal of the media,
500 μL of DMSO was added to the wells to dissolve the
converted dye. The solution (150 μL) from each sample
was transferred to 96-well plates and the optical density
(O.D.) was measured with an ELISA reader (BIORAD,
Munich, Germany) at an absorbance of 490 nm.
2.5.2. Cel l Imaging
Cell morphology study on PM-BC-3 scaffold was carried
out by FE-SEM. After pre-soaking with DMEM over-
night, PM-BC-3 samples (Ф10 × 1 mm) were incubated
with MDA-MB-231 cells at a density of 2 × 105 cell/mL
in 24-well plates for 14 days at 37˚C in 5% CO2. After 14
days incubation, the cell-scaffold samples were rinsed
twice with PBS and fixed using 4% glutaraldehyde for 12
h, and then dehydrated through gradient concentration of
ethanol a series of graded alcohols (40%, 50%, 60%,
70%, 80%, 90% and 100%), and air-dried. Finally, the
samples were sputter-coated with a layer of gold as de-
scribed above and observed by SEM to analyze cell ad-
hesion and morphology.
2.5.3. Hist ol o g i cal Anal ysis
After 3, 7, 14, 21, and 28 days culture, the cell-scaffold
constructs were washed with ice-cold normal saline
(0.9% NaCl), cut transversely into thin slices (5 μm), and
then fixed into 10% neutral-buffered formaldehyde for
24 h. The tissues were then transferred into 70% ethyl
alcohol, processed, and embedded in paraffin wax. The
sections with cells were stained with hematoxylin and
eosin (H&E) for histological examination using a Zeiss
Axioplan-2 fluorescence light microscope (Carl Zeiss,
Inc., NY).
2.6. Statistical Analysis
All experiments were performed in triplicate unless oth-
erwise stated. Statistical analysis of data was performed
using an SPSS system. All data were presented as mean
values ± standard deviation (SD). Results with p-values
of <0.05 were considered statistically significant.
3. Results and Discussion
3.1. Morphology of the PM-BC Scaffolds
Figure 2 shows the optical and SEM images of three
PM-BC scaffolds. Note that aligned channels were cre-

A Novel in Vitro Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of
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320
Figure 2. Optical (a)-(c) and SEM (d)-(i) photos of PM-BC-
1 (a), (d), (g), PM-BC-2 (b), (e), (h), and PM-BC-3 (c), (f),
(i).
ated in the BC scaffolds by computer-aided laser pat-
terning technique and that both the density and diameter
of the patterned macropores were controllable (Figures
2(a)-(c)). As displayed in Figures 2(d)-(f), the obtained
pore diameter was about 170, 169, and 315 μm for PM-
BC-1, PM-BC-2, and PM-BC-3, respectively, which was
15 - 20 μm larger than the designed values. High magni-
fication SEM images (Figures 2(g)-(i)) revealed that the
wall of each macropore was smooth without any debris
that was often observed for UV and femtosecond laser
micromachining. Figures 2(g)-(i) also showed that the
wall of each macropore was porous, consisting of nano-
fibers and micropores (<100 μm as defined in the litera-
ture [33,36]) and nanopores (<100 nm) (see Section 3.2),
which was actually the intrinsic characteristics of pristine
BC [44]. This structural feature was very favorable to the
cell functions since porous wall favored cell functions
[45] while compact pore walls hindered metabolite diffu-
sion thus restricting cell proliferation and migration in-
side 3D scaffolds [46]. In other words, when cells were
seeded onto the internal porous matrix of PM-BC scaf-
folds, the inner porous wall was believed to be able to
keep deeply embedded cells supplied with nutrients [47].
3.2. Pore Size Distribution
Scaffold pore structure included porosity, pore size, pore
geometry, pore branching, pore connectivity and pore
orientation [33]. Though SEM revealed the co-existence
of macropores (>100 μm), micropores, and nanopores in
the PM-BC scaffolds, porosity and pore size distribution
could not be obtained by SEM. In this work, mercury
intrusion porosimeter was used to obtain porosity and
pore size and its distribution of the pristine BC, and the
pore volume percentage and porosity of three PM-BC
scaffolds were thus obtained. The results are listed in
Table 2. As could be seen from this table, the dominant
pores in the pristine BC scaffold were in the range of 10
to 100 µm (mostly at ca. 20 µm) and a few nanopores
with a diameter of 4 - 6 nm were also observed. This
small pore size might interpret the poor cell adhesion,
proliferation, and non-infiltration to the inner side of the
pristine BC scaffolds reported by Szot et al. [32]. It was
noted that a significantly different pore structure was
observed for PM-BC scaffolds. Three PM-BC scaffolds
contained macropores in addition to nanopores and mi-
cropores. Though all the four samples showed hierarchi-
cal pore structure, the three PM-BC samples showed a
much wider pore diameter distribution because of the
existence of macropores, which would be beneficial to
the culture of cells as macropores were favorable to cell
migration and particularly for cell infiltration to the core
of scaffolds.
It was reported that a combination of large and small
pores was necessary for tissue engineering scaffolds be-
cause the large pores acted as the passage of nutrients
and wastes and provided the areas where different cell-
types can be deposited, while the small pores favored to
provide correct signals to encourage differentiation and
to pattern cells as they differentiate into an organized tis-
sue [47]. A study on multi-layered 3D scaffolds consist-
ing of microchannels (250 μm) and inner smaller pores
(<10 μm) confirmed the notion [48]. As cancer cells usu-
ally had a larger size than normal tissue cells, the exis-
tence of large pores would be mandatory for cancer cell
metastasis in addition to small pores.
3.3. Tensile Properties
Figure 3 shows the tensile strength and modulus, and
strain at break of three PM-BC and pristine BC samples.
As expected, all three PM-BC exhibited significantly
lower tensile strength (p < 0.05 in all cases) than pristine
BC and PM-BC-1 had significantly lower tensile mo-
dulus and strain (p < 0.05) than pristine BC. However,
the differences in tensile modulus and strain between
pristine BC and PM-BC-2 and PM-BC-3 were not sig-
nificant (p > 0.05), indicating that tensile strength was
more sensitive to macropores as compared to tensile mo-
dulus and strain. The significantly lower tensile modulus
and strain of PM-BC-1 than PM-BC-2, PM-BC-3, and
pristine BC was simply due to its higher pore density in
comparison to other two PM-BC samples (comparing
pore distance listed in Table 1), suggesting that the mo-
dulus and strain of porous scaffolds were not merely con-
trolled by pore volume, instead they were also relevant to
pore density.
Although about 12% reduction in tensile strength was

A Novel in Vitro Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of
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321
Table 2. Comparisons of pore volume percentage (%) and porosity (%) of various BC scaffolds.
Micropores (%)
Samples Nanopores (%)
<100 nm 1 - 10 μm 10 - 100 μm
Macropores (%)
>100 μm Porosity (%)
Pristine BC 1.36 25.19 66.81 0.00 92.05
PM-BC-1 1.33 23.88 65.95 1.28 92.16
PM-BC-2 1.30 24.65 65.38 2.14 92.23
PM-BC-3 1.30 24.08 63.88 4.38 92.44
folds still very competitive for tissue engineering and
tumor engineering scaffolds in terms of mechanical per-
formances.
Pristine BCPM-BC-1PM-BC-2PM-BC-3
0.0
0.2
0.4
0.6
Tensile strength (MPa)
(a) +
Many previous studies on bone tissue engineering
have suggested a need for pore size >300 µm for bone
formation and vascularisation [40,45,50]. Considering
cancer cells are usually larger in size than normal cells,
PM-BC-3 with 315 µm pore size was believed to be a
more suitable candidate for cancer cell culture in com-
parison to PM-BC-1 and PM-BC-2. Furthermore, PM-
BC-3 demonstrated higher tensile properties than PM-
BC-1 and comparable to PM-BC-2. Therefore, PM-BC-3
was used in the subsequent cell studies.
Pristine BCPM-BC-1PM-BC-2PM-BC-3
0
2
4
6
8
10
12
14
16
Tensile modulus (MPa)
(b)
#
##
3.4. Cell Viability and Proliferation
The MTT assay result shown in Figure 4 demonstrated
that cancer cells were viable and the proliferation of
MDA-MB-231 cell line was robust, keeping a constant
rate during 7 days culture. However, limited viability and
proliferation were observed by Szot et al. when cancer
cells were cultured on the pristine BC without introduc-
tion of macropores [32]. The difference in cell prolifera-
tion suggested that the pore structure and size of the tu-
mor engineering scaffolds were crucial to the culture of
cancer cells.
0
1
2
3
4
5
6
7
8
9
Elongation (%)
Pristine BCPM-BC-1PM-BC-2 PM-BC-3
(c)
***
3.5. Morphology of Cells on the PM-BC-3
Scaffold
Previous study by Szot et al. demonstrated that cancer
cells cultured on BC were not spread out across the sur-
face of BC scaffolds due to the absence of manufactured
porosity [32]. In this study, adhesion and morphology of
the MDA-MB-231 cells after 7 days seeding on the PM-
BC-3 scaffold were characterized by SEM and the results
are shown in Figure 5. SEM demonstrated that cancer
cells were attached to the scaffold. Note that individual
cancer cell and cancer cell aggregates attached and spread
throughout the surface of the PM-BC-3 scaffold and
many cells connected to the neighboring cells as shown
in Figures 5(a) and (b). Figures 5(c) and (d) demon-
strated a wide spread and tight attachment to the scaffold.
Furthermore, the MDA-MB-231 cells on the PM-BC
Figure 3. Tensile strength (a), tensile modulus (b), and strain
at break (c) of various materials (p+, p#, and p* < 0.05; p##
and p** > 0.05).
observed because of the creation of macropores, the ten-
sile strength value of these PM-BC samples was still
much higher than other natural biomaterials. For instance,
it was 40-fold higher than hyaluronan-collagen scaffolds
(10 - 15 kPa) [49]. Tensile testing indicated that the pre-
sence of macropores did not sharply decrease the tensile
properties of BC samples, which made the PM-BC scaf-

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01234567
0.0
0.2
0.4
0.6
0.8
1.0
1.2
O.D. (490 nm)
Culture time (day)
Figure 4. Proliferation of MDA-MB-231 cell line cultured
on the PM-BC scaffold by MTT assay.
Figure 5. SEM images of MDA-MB-231 cells on the PM-BC
scaffold after 2 weeks culture ((a)-(d) showing varying mag-
nification).
scaffold kept their normal morphology of roughly rounded
shape (3D image: oblate spheroid), which was similar to
that seen in cells grown on chitosan-alginate (CA) 3D
scaffolds reported by Kievit et al. who declared that cells
in solid tumors exhibit similar morphology and thus con-
cluded that the CA scaffolds could provide a growth en-
vironment that promoted the formation of solid tumor-
like cells [11]. More importantly, a large number of pro-
truded pseudopodiums were formed that bonded to the
scaffold (see arrows in Figures 5(c) and (d)), indicating
a strong adhesion of cancer cells to the scaffold. It was
striking to note that multilayered cancer cells were ob-
served on the surface of the PM-BC scaffold (circle in
Figure 5(d)) and moreover, cancer cells in the basal layer
formed clusters (ellipse in Figure 5(d)), which could
never be found for conventional 2D culture due to its
limited space. The SEM results suggested that the PM-
BC-3 scaffold strongly supported the adhesion, spreading,
proliferation, and differentiation of the MDA-MB-231
cell line.
3.6. Histological Observation
SEM observation was not able to provide any informa-
tion inside the scaffold. In this study, histological obser-
vation was performed to evaluate the cells distribution
inside the PM-BC scaffold upon culture for varying pe-
riods up to 4 weeks. The H&E staining results are shown
in Figure 6. Szot and co-workers reported that no cancer
cell infiltration was observed after 7 days culture on BC
[32] while the results of the present study were totally
different. As clearly seen in Figure 6(A), the distribution
of the MDA-MB-231 cells within the PM-BC-3 scaffold
was observed, suggesting cells migration into the macro-
porous scaffold and further formed clusters in some areas
(circles in Figures 6(A-b) and (A-c)) even after only 3
days culture. After 7 days culture (Figure 6(B)), the cells
spread and grew along the walls of the macropores (see
arrows in Figure 6(B)) and more cell clusters were found
(see circle in Figure 6(B-c)), indicating successive growth
and proliferation inside the PM-BC-3 scaffold. Figures
6(A) to (E) clearly showed an increased cell density and
increased number of clusters (Figure 6(E-c) as a repre-
sentative) with culture time, indicating that cancer cells
experienced robust proliferation, in-growth, and differen-
tiation inside the PM-BC-3 scaffold. These findings sug-
gested that cancer cells could penetrate into the core of
the PM-BC-3 scaffold due to the presence of macropores.
The histological observation together with the SEM and
MTT results was sufficient to verify that the PM-BC-3
scaffold was able to promote the adhesion, in-growth,
proliferation, and differentiation of the MDA-MB-231
cells and thus this macroporous material could be a novel
scaffold for the in vitro culture of cancer cells.
4. Conclusion
Patterned macroporous BC scaffolds were successfully
prepared by a one-step direct perforation in BC hy-
drogels by using infrared laser micromachining tech-
nique. The PM-BC scaffolds with different pore size and
pore density could be obtained. Mechanical tests re-
vealed that the PM-BC scaffolds could maintain 88 per-
cent tensile strength of the pristine BC scaffolds and in-
significant difference in tensile modulus and strain at
break were observed between the pristine BC and two
PM-BC samples with pore distance of 1.0 mm. All three
PM-BC scaffolds prepared in this work showed hierar-
chical pore size distribution from several nanometers to
over a hundred microns, but the PM-BC-3 scaffold showed
the largest pore size (315 µm) and porosity and the wid-
est pore size distribution among the three macroporous
BC scaffolds. It was concluded that, similar to UV and
femtosecond lasers which were suitable for metals and
polymers, the infrared laser was an excellent tool for

A Novel in Vitro Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of
Breast Cancer Cells
323
Figure 6. Histological analysis of MDA-MB-231 cell line grown in PM-BC-3 scaffold 3 (A), 7 (B), 14 (C), 21 (D) and 28 (E)
days post-seeding.
macropatterning BC hydrogels as a rapid and chemical
free process and thus showed a great potential in macro-
machining other hydrogels. Results of the cell prolifera-
tion assay with almost linear growth suggested that the
PM-BC-3 scaffold could support the growth and prolif-
eration of MDA-MB-231 cell line. SEM indicated that
the MDA-MB-231 cells on the PM-BC-3 scaffold kept
their normal morphology and that multilayered growth
was noted. Both SEM and histological analysis revealed
the existence of cell clusters. Additionally, histological
analysis revealed that cells attached and infiltrated into
the PM-BC-3 scaffold 3 days post-seeding. Cell studies
with the PM-BC-3 scaffold showed that this laser-pat-
terned macroporous scaffold exhibited good cell com-
patibility, could promote cell adhesion and spread on the
scaffold surface, and cells were able to proliferate within
the scaffold, indicating the satisfactory biocompatibility
of the macroporous scaffold.
5. Acknowledgements
This work is supported by the National Natural Science
Foundation of China (Grants No. 51172158, 81272358,
30930038 and 81200663) and the Science and Technol-
ogy Support Program of Tianjin (Grant No. 11ZCKFSY-
01700).

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Breast Cancer Cells
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