ARTICLE IN PRESS

Biomaterials 27 (2006) 2141–2149

www.elsevier.com/locate/biomaterials

Mechanical properties of bacterial cellulose and interactions

with smooth muscle cells
Henrik Bäckdahla,b, Gisela Heleniusb, Aase Bodina, Ulf Nannmarkc, Bengt R. Johanssonc,
Bo Risbergb, Paul Gatenholma,
a
Department of Chemical and Biological Engineering, Biopolymer Technology, Chalmers University of Technology, SE-412 96 Göteborg, Sweden
b
Department of Surgery, Vascular Engineering Centre, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden
c
Department of Anatomy and Cell Biology, Electron Microscopy Unit, Sahlgrenska University Hospital, Gothenburg University, SE-405 30 Göteborg, Sweden
Received 9 June 2005; accepted 31 October 2005
Available online 28 November 2005

Abstract

Tissue engineered blood vessels (TEBV) represent an attractive approach for overcoming reconstructive problems associated with
vascular diseases by providing small calibre vascular grafts. The aim of this study has been to evaluate a novel biomaterial, bacterial
cellulose (BC), as a potential scaffold for TEBV. The morphology of the BC pellicle grown in static culture was investigated with SEM.
Mechanical properties of BC were measured in Krebs solution and compared with the properties of porcine carotid arteries and ePTFE
grafts. Attachment, proliferation and ingrowth of human smooth muscle cells (SMC) on the BC were analysed in vitro. The BC pellicle
had an asymmetric structure composed of a fine network of nanofibrils similar to a collagen network. The shape of the stress–strain
response of BC is reminiscent of the stress-strain response of the carotid artery, most probably due to the similarity in architecture of the
nanofibrill networks. SMC adhered to and proliferated on the BC pellicle; an ingrowth of up to 40 mm was seen after 2 weeks of culture.
BC exhibit attractive properties for use in future TEBV.
r 2005 Elsevier Ltd. All rights reserved.

Keywords: Bacterial Cellulose; Scaffold; Smooth muscle cell; Tissue engineering blood vessels

1. Introduction bovine aortic cells in a collagen-gel matrix to construct

Cardiovascular disease is the dominant cause of mor-
bidity and mortality in the adult population. Today it is
possible to replace large arteries with synthetic grafts made
from polyester or expanded-polytetraflourethylene
(ePTFE) [1–3]. These materials are however not suitable
as a replacement in small caliber grafts, o6 mm in
diameter, due to thrombosis formation and occlusion
[4–6]. New materials that are not thrombogenic and have
mechanical properties that mimic the native vessel are
therefore required.
Tissue engineering of blood vessels (TEBV) is emerging
as a means of producing replacement vessels. Weinberg
and Bell became pioneers in this field when they produced a
completely biological TEBV in 1986. They embedded
Corresponding author. Tel.: +46 31 772 34 07; fax: +46 31 772 34 18.
E-mail address: Paul.Gatenholm@chem.chalmers.se (P. Gatenholm).
the three vascular wall layers. Extensive research by many
groups has focused on improving the collagen based
constructs [7–11]. L’Heureux et al. constructed a vessel of
vascular cell sheets. They rolled a smooth muscle cell
(SMC) sheet around a mandrel of ePTFE to form a tubular
media layer [12]. Cell seeded degradable polyglycolic acid
(PGA), poly(L-lactic acid) (PLLA) and co-polymers thereof
have been widely used as scaffolds [13–15]. Niklason et al.
developed a tissue engineered artery constructed from a
PGA mesh seeded with SMCs in 1999 [14]. In spite of the
intensive research in this area no completely biocompatible
replacement TEBV suited for small diameter vessel
replacement is currently in clinical use.
Bacterial cellulose (BC) is a polysaccharide that is
excreted extracellularly by the Acetobacter xylinum bacteria
into long non-aggregated nanofibrils [16]. The BC displays
many unique properties including high mechanical
strength, high water content, high crystallinity and an

0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2005.10.026
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2142 H. Bäckdahl et al. / Biomaterials 27 (2006) 2141–2149
ultra-fine highly pure nanofibril network structure [17–19].
Acetobacter xylinum constructs a pellicle of BC that has a
dense surface side and a gelatinous layer on the opposite
side. BC has been used in a micro vessel endoprothesis [20]
and as a temporary skin substitute [21]. The material has
also been investigated as a scaffold for tissue engineering of
cartilage [22]. To our knowledge, BC is not widely used or
investigated for tissue engineering applications. The aim of
the present study was therefore to investigate the mechan-
ical properties of BC and study the SMC-BC interactions
to explore whether BC can be suitable for use in TEBV.
2. Methods and materials
2.1. Bacterial cellulose
BC was grown static in corn steep liquid media, described elsewhere
[23], using Roux flasks (working volumes 100 ml) at 30 1C for 3 days,
giving a pellicle of 3 mm. The strain used for the synthesis was Acetobacter
xylinum subsp. sucrofermentas BPR2001. The strain was purchased from
the American Type Culture Collection with the code ATCCXXXX. The
BC was purified by boiling in 0.1 M NaOH at 60 1C for 4 h and thereafter
by repeated boiling in MilliporeTM water. The material was steam
sterilized (1 bar, 120 1C) for 20 min and kept refrigerated until use.
2.2. Mechanical properties of bacterial cellulose, porcine carotid
artery and ePTFE
Tensile tests were made with a small tensile tester rebuilt to make
possible tensile tests, see Fig. 1 (specially designed tensile tester,
Universität Essen), in aqueous conditions.
Experiments were run at 0.25 mm/s cross head speed. Tests were done
in Krebs solution [24] at a temperature of 3771 1C. Ten BC rings, with an
outer diameter of 10 mm, inner diameter of 5 mm and cylinder length of
2.6 mm, were punched out from a cultured BC pellicle described above.
The BC samples were allowed to condition in Krebs solution for 24 h prior
to tensile testing. A 7–9 cm long fragment of the porcine carotid artery,
Swedish landrace, was removed immediately post mortem and placed in
chilled Krebs solution [24]. Ten ring shaped sections were made from the
artery, each 2 mm long. The artery rings were allowed to condition in
37 1C Krebs for 1 h prior to tensile testing. Ten 2-mm long rings of ePTFE
tube with an inner diameter of 3 mm and a thickness of 0.5 mm were used
as a second reference material. Stress (s) is calculated by F =A where A is
the area measured as in Fig. 1B and F is force in Newton. Strain (e) is
calculated by DL=L0 where DL is exerted extension from starting point L0.
Fig. 1. (A) Principle set-up of tensile test procedure, starting position.
Rings were stretched in the vertical direction. (B) Cross section area (A) of
samples used in tensile tests experiments.
2.3. Preparation of cells and cell culture
SMCs were isolated from healthy parts of human saphenous veins or
arteries. Veins were either spare parts from coronary bypass operations or
were taken from patients undergoing surgery for varicose veins. Arteries
were splenic and superior mesenteric arteries from patients undergoing
either splenectomy for hypersplenism or resection for colon carcinoma.
The Ethics Committee at Gothenburg University approved the project.
SMCs were isolated using an explant technique [25] and the cells were
subcultured in Dulbeco’s Modified Eagle Medium, DMEM (PAA
Laboratories GmbH), supplemented with 20% fetal bovine serum,
100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate (PAA Labora-
tories GmbH), 0.1 mM non-essential amino acids (GibcoBRL/Life-
Technologies Ltd, Palsley, Scotland, UK) and 2 mM L-glutamine. After
the first passage, SMC were cultured in DMEM as described above but
with 10% fetal bovine serum. Antibodies directed against a-actin (DAKO
A/S, Glostrup, Denmark) confirmed the identity of smooth muscle cells.
SMC were negative for endothelial cell markers, von Willebrand factor
(DAKO A/S) and PECAM-1 (Santa Cruz Biotechnology Inc., Santa Cruz,
USA), and were also recognized by their typical ‘‘hill and valley’’ pattern.
SMC cultures were incubated in a humidified atmosphere of 95% air/
5% CO2 and a temperature of 37 1C. The culture medium was changed
once a week. All cells tested negative for mycoplasma contamination. The
cells were subcultured and used in passage 5–7.
2.4. Cell attachment
The Alamar Blue assayTM (Serotec Ltd Scandinavia, Hamar, Norway)
was used to measure the number of cells that adhered to the BC. The
Alamar Blue assayTM is designed to quantify the proliferation and
viability of cells [26,27]. In the attachment experiments, the number of
viable cells corresponds to the number of adherent cells. A cell
concentration of 4000 cells/cm2 was used for the attachment experiments.
The SMCs were seeded on round pieces of BC (2 mm thick) or on cell
culture treated polystyrene that was used as a reference material. The cells
were allowed to attach to the materials for 24 h. After this incubation, the
pieces of BC were moved to new wells to avoid false signals from cells on
the bottom of the well. All cell culture media were removed and new
media+10% Alamar Blue reagent were added. After 5 h incubation with
the Alamar Blue reagent, samples of 100 ml were pipetted into a 96 well
plate and the fluorescence was measured at excitation wavelength 544 nm
and emission wavelength 590 nm in a spectrofluorometer (Spectramax
GeminiXS, Molecular Devices, Sunnyvale, CA, USA). To avoid misread-
ing the autofluorescence of the materials was subtracted from the test
results.
2.5. Cell proliferation
The attachment measurement gave the starting point of the prolifera-
tion measurements, 5 h. The experiment was continued after the
attachment measurement and sequential samples were taken after 1, 3, 5
and 7 days. Each sample was evaluated with the same equipment as
described under cell attachment.
2.6. Cell ingrowth
A migration chamber and an attractant were used to stimulate the cells
to grow into the BC. The migration chamber consisted of cell culture
inserts with a membrane pore size of 8.0 mm in a 12 well plate (BD
FalconTM, BD, NJ, USA). Pieces of BC were placed in the inserts and
arterial SMCs (10 000 cells/cm2) were seeded on top of the BC. To attract
the cells to migrate into the material, 12 ng/ml of platelet derived growth
factor-BB (PDGF-BB) [28] (AMS Biotechnology Ltd, Oxon, UK) were
added to the cell culture medium (DMEM as described above) in the wells.
The cell cultures were incubated for 1, 2 and 3 weeks and the culture
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H. Bäckdahl et al. / Biomaterials 27 (2006) 2141–2149
medium was changed twice weekly. After conclusion of the experiments,
the specimens were fixed and processed for electron microscopy.
2.7. Confocal microscopy
For analysis of cell morphology, the cells were fixed in 3.7%
formaldehyde and permeabilized in 0.2% Triton X-100 in PBS. To
visualize f-actin, cells were stained with phalloidin conjugated with
Alexa Fluor 546 (Molecular Probes Inc., Eugen, OR, USA). The cell
nuclei were counter stained with 40 ,6-diamidino-2-phenylindole,
dihydrochloride, DAPI (Molecular Probes Inc.). The specimens were
mounted in SlowfadeTM Antifade mounting medium (Molecular Probes
Inc.). The cells were analysed with a laser scanning confocal microscope,
Leica TCS SP2 AOBS (Leica Microsystems, Heidelberg GmbH,
Germany).
2.8. Electron microscopy
Comparisons were made between BC and collagen fibres from
umbilical cord and cell morphology and ingrowth were studied with
electron microscopy, TEM (LEO 912 AB Omega) and SEM (LEO 982
Gemini field emission SEM). BC samples were quenched in liquid nitrogen
and freeze dried. BC samples with SMCs were fixed overnight by
immersion in a mixture of 2% paraformaldehyde and 2.5% glutaralde-
hyde in 0.05 M Na cacodylate buffer (pH 7.2).
Postfixation was done with 1% OsO4 and 1% potassium ferrocyanide
in 0.1 M cacodylate for 2 h at 4 1C, followed by en bloc staining with 1%
uranyl acetate in H2O for 1 h. Specimens were then dehydrated in a graded
series of ethanol and infiltrated with epoxy resin (Agar 100, Agar Scientific
Ltd., Stanstead, UK), followed by curing by heat. Semi-thin sections
(0.7 mm) were cut and examined in a light microscope in order to orient
specimens correctly before cutting ultra-thin sections (50–60 nm) using a
Reichert ultramicrotome equipped with a diamond knife. Sections were
collected on copper grids and counter stained with lead citrate and uranyl
acetate before examination in a Zeiss 912AB fully digitized electron
microscope [29]. Digital images were taken with a Megaview III camera
(SIS, Munster, Germany).
2.9. Statistics
In the analysis of the mechanical properties, the statistical significance
was tested by an unpaired Student t-test. A non-parametrical Wilcoxon
test was used to check whether the data were normally distributed. There
were no differences in the results between the two tests. Po0.05 was
considered significant.
Fig. 2. Visualization of BC pellicle side differences, SEM pictures of quenched and frieze dried BC pellicle (A) denser surface side that was formed at the
interface between air and culture medium (B) porous opposite side.
2143
3. Results
3.1. Characterization of BC
Statically cultured BC formed a pellicle. This pellicle was
composed of a small amount of nanofibrils holding about
99% water. The network of fine, highly entangled
nanofibrils with dimensions of approximately 100 nm was
seen in SEM. The nanofibrils’ network was much denser
close to the medium/air transition zone (Fig. 2A) than on
the opposite side (Fig. 2B).
The denser surface side had a small amount of
completely dense areas separated by more porous areas,
and the visualization depth was shorter than on the porous
opposite side.
3.2. Mechanical properties
Tensile testing was done in conditioned Krebs solution
with a cross head speed of 0.25 mm/s. Three materials were
investigated: rings of BC, porcine carotid artery (PCA) and
ePTFE. Fig. 3 gives the mean values (m) and standard
deviations (s) of the tested materials’ tensile properties
(n ¼ 10); tensile strength (smax (MPa)), deformation at
break (emax (%)) and Young’s modulus (E (MPa)) were
measured.
The BC rings were far more similar to PCA than ePTFE.
A PCA significantly exceeds the BC pellicle in both stress at
break, strain at break and Young’s modulus. The
difference in Young’s modulus was not as high as for the
other parameters, 1.0470.31 MPa as compared to
0.5970.12 MPa.
Fig. 4A shows a typical stress-strain response of the BC
material. The strain induced strength development was
seen first after elongation above 35%. After this extension
the sample showed linear elastic behaviour. We did not,
however, see linear elastic behaviour at low deformation.
The nanofibril rearrangement at deformation is visualized
in Fig. 4B, where the nanofibrils are aggregated into thicker
fibre clusters upon extension. The deformation behaviour
of ePTFE is visualized in Fig. 4C. The material showed
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2144 H. Bäckdahl et al. / Biomaterials 27 (2006) 2141–2149

Strain at break
800

Tensile test results

25
600
Stress at break and Young´s modulus

20

%
400

15
200
MPa

10
0

5
Bacterial cellulose
Porcine carotid artery
ePTFE
0
Stress at break Young´s modulus

Fig. 3. Mean7standard deviation of tensile tests preformed in Krebs solution. The ePTFE had significantly higher values than both BC and the porcine
carotid artery (PCA) in all tests. BC was more similar to the PCA than ePTFE when looking at stress at break and Young’s modulus. When comparing the
data for strain at break its important to also look at Fig. 3, there it can be seen that ePTFE has a yield point at a strain of 55% and then starts to deform
irreversibly, the other materials don’t have this phenomenon. The Young’s modulus values of BC was close to that of PCA, but the difference was
significant (Po0.05).

elastic deformation up to an elongation of 55%, where a
yield point was seen, which was followed by irreversible
deformation. Porcine carotid artery (Fig. 4D) showed a
similar deformation appearance as BC, with the exception
of the
300% elongation required for strain induced
strength development.
occurred rapidly and had reduced all Alamar BlueTM in the
medium after 3 days. No significant difference in the
proliferation rate of SMCs on the compact and the porous
sides of the BC was detected. Proliferation of SMC on BC
was slower than on the reference polystyrene surface
(Fig. 6).

3.3. Comparison between collagen and BC 3.5. Cell morphology and ingrowth

Microscopically, the bacterial cellulose nanofibrils had a
shape similar to that of the collagen nanofibrils (Fig. 2),
both with a dimension of 100 nm in diameter.
No cells were removed from the collagen (Fig. 5A), the
hollow spaces seen inbetween the collagen fibres were filled
with large quantities of water and matrix in vivo. However,
during fixation, dehydration and hexametyldisilazan pre-
paration, water and substances dissolved in the water
disappear. The size of Acetobacter xylinum is much smaller
than the spaces in between the nanofibrils in the BC
pellicle.
3.4. Cell attachment and proliferation
In these experiments, the number of viable cells was
parallel to the number of adherent cells. With the Alamar
Blue assayTM, cell attachment measured at 5 h was
significantly lower on BC than on polystyrene (Fig. 6).
There were no differences in the attachment of SMCs on
the compact and porous sides of BC.
With the Alamar Blue AssayTM, the proliferation was
measured sequentially on the same cells for 7 days. Samples
of the cell culture medium were taken after 5, 24, 72, 120
and 168 h. Proliferation of SMCs on the reference material
The morphology of the SMCs was studied with laser
confocal scanning microscopy. There was no obvious
difference in the morphology of the cells cultured on the
compact side and the porous side. SMCs cultured on BC
for 3 days showed spreading similar to that of the cells
cultured on polystyrene. The cells on the BC did not have
the distinct f-actin stress fibres seen in the cells at on the
reference material in Fig. 7. The surface of culture plastic is
completely smooth while the cells on BC experienced a 3D
structure. The BC is a hydrogel with nanofibrils while a
culture plastic is solid. These two parameters greatly
influence how the cell nuclei and actin is represented in
the picture.
TEM pictures revealed that cells could push the
nanofibrils aside when they migrated into the cellulose
nanofibril network, as shown in Fig. 8. BC fibrils are seen
as small white/grey dots in the TEM pictures (Figs. 8B, 9A
and B). The concentration of nanofibrils seen above the
migrated cell (Fig. 8B) was much higher than in other parts
of the sample.
The cells on the compact side appeared to be thin and
elongated while the cells on the porous side were more
rounded. SMCs on the porous side of the BC had migrated
up to 10 mm into the fibrous network after 2 weeks of
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H. Bäckdahl et al. / Biomaterials 27 (2006) 2141–2149 2145

Stress/Strain diagram of BC rings

0.18
0.16
0.14
0.12
Stress (MPa)

0.10
0.08
0.06
0.04
0.02
0.00
0 20 40 60 80 100 120 140
(A) Strain (%) (B)

Stress/Strain diagram of ePTFE Stress/Strain diagram of porcine carotid artery

14 1.0

12
0.8
10
Stress (MPa) 0.6
Stress (MPa)

6 0.4

4
0.2
2
0.0
0

0 200 400 600 800 0 100 200 300 400 500 600 700 800
(C) Strain (%) (D) Strain (%)

Fig. 4. Stress/strain diagrams of tensile test results made with 0.25 mm/s cross head speed on: (A) BC rings had similar shape to the porcine carotid artery
(PCA) (B) SEM picture of the BC nanofibril network close to fracture. Fibres had rearranged into thick fibre aggregates. (C) ePFTE rings results differ a
lot, both in stress at break and shape, from BC and the carotid artery. (D) Diagram showing results from porcine carotid artery, elongation of PCA before
stress increase is greater than for BC rings,
300% compared to
35%.

Fig. 5. Visual comparison of collagen and BC showed a similar fibre structure. SEM pictures of samples prepared by Karnovsky fixation. (A) Collagen
from umbilical cord and (B) bacterial cellulose produced by Acetobacter xylinum.

culture while no cell ingrowth was detected from the
compact side. When the experiments were made with
PDGF-BB, however, the ingrowth on both sides was
higher. Fig. 9A shows the migration of an SMC into the
BC nanofibril network from the porous side after 1 week
in culture with PDGF-BB. The maximum ingrowth
distance observed after 1 week was 20 mm. An ingrowth
distance up to 40 mm could be seen after 2 weeks in culture.
The SMCs could now be found at different depths
(Fig. 9B), as compared to only one depth after one
week. The more compact side showed only a slight
ingrowth of 1–5 mm.
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Adhesion and proliferation of SMC 4. Discussion

4000

The present study evaluated the potential of bacterial

3000 cellulose to function as a scaffold for tissue engineered
blood vessels in vitro.
Fluorescence

2000 SMCs are the major cell type in tunica media and are
widely used in the TEBV field [12,14]. The BC pellicle holds
99% water, and this allows room for cell ingrowth and
1000
proliferation. SMCs attached to both sides of BC. They
were viable on the material up to 8 weeks. There was no
0 BC compact side difference in the number of cells that adhered to the
BC porous side
Culture plastics compact and the porous sides. In the Alamar Blue assay,
the cells’ ability to reduce a redox indicator corresponds to
0 20 40 60 80 100 120 140 160 180 the viability and thereby also attachment, since only the
Culture time (hours) attached cells are viable. The cell metabolism and thereby
Fig. 6. Alamar BlueTM assay results showed that SMC adhere, after 5 h of the ability to reduce a redox indicator could vary
culture, to both sides of bacterial cellulose and the reference material, depending on the surface properties of the material to
polystyrene. There were no significant differences between the two sides of which it is attached. It is important to take both surface
cellulose, while the culture plastic surface had a significantly higher chemistry and topography into consideration. A relevant
attachment. The cells proliferated better on culture plastics than on BC,
they had consumed all Alamar Blue substances after 72 h. SMC
comparison is cells cultured on a collagen gel in that both
proliferated similarly on both sides of the nanofibril BC network. collagen and BC fibrils are fibrous hydrogels. Fibroblasts

Fig. 7. Laser scanning confocal images of SMC cultured on the porous side of BC (A) and polystyrene (B) f-actin stress fibres are stained red with
phalloidins conjugated with Alexa 546. Cell nuclei are counter stained with DAPI (blue). In A, the f-actin stress fibres are less distinct compared to those in
B. Cell nuclei in A are more irregular compared to those in B. The degree of cell spreading is similar in A and B.

Fig. 8. (A) SEM picture of SMC migrating into the BC pellicle, arrows pointing on it. The fibre network can be seen above the cells. (B) TEM picture of a
migrating SMC, BC nanofibrils is seen as small white/grey dots indicated by arrows. A surface cell is seen to the right in picture. The concentration of
white/grey dots is much higher inside the ellipse then outside of it, indicating that the cells push the BC nanofibril network aside when migrating into the
material.
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H. Bäckdahl et al. / Biomaterials 27 (2006) 2141–2149
Fig. 9. TEM pictures of SMC ingrowth from the porous side, bacterial cellulose nanofibrils are seen as small white/grey dots. (A) 1 week, surface SMC cell
seen to the left in picture. A few cells were found to have migrated into the material, BC nanofibril network is seen between surface and ingrown cell,
indicated by arrow. (B) 2 weeks, surface cell seen in the upper right corner of the picture. A greater number of cells were found in the BC pellicle and at
different depths (1, 2 and 3).
synthesize less collagen when cultured on collagen hydro-
gels as compared to when they are cultured on tissue
culture plastics [30]. In another study, the collagen
synthesis in SMCs cultured in collagen was only 20% of
that in culture on cell culture plastics [31]. Thus it is likely
that both the protein synthesis and metabolism differ
between cells cultured on fibrous hydrogels and smooth
polystyrene. This might explain the differences in attach-
ment between the BC surface and culture plastics seen in
Fig. 6.
The major advantage of the Alamar Blue assay is that it
is possible to follow the same cells during the culture time.
With other methods, the cells must be sacrificed at each
time point. The Alamar Blue assay was therefore chosen
here to evaluate proliferation. SMCs proliferated equally
on both sides of the BC. The proliferation rate was much
lower in comparison with the reference material. That can
be explained by the discussion above concerning attach-
ment. It is however relevant to discuss whether a
proliferative SMC population is desirable in a tissue
engineered construct. Initially, during the in vitro culture
time, proliferation is necessary to achieve a high number of
cells. However, when implanted into a host, a quiescent,
differentiated and contractile phenotype is preferable [32].
Vascular smooth muscle cells cultured in vitro typically
revert from a contractile phenotype to a synthetic
phenotype. Pulsatile strain and shear stress stimulate
vascular smooth muscle tissues development and induce
the vascular SMC to retain the differentiated phenotype in
vascular smooth muscle engineering in vitro. This is
measured by investigating the levels of smooth muscle a-
actin compared to static cultured tissue [33]. This will be
further studied when culturing SMC on a tubular BC
scaffold.
The morphology of bacterial cellulose is similar to that
of asymmetric membrane with one more compact side and
another more porous side. Large differences in SMC
morphology were seen after one week in culture depending
on which side of the BC pellicle the cells were cultured. The
cell morphology was the same for cultures with and
2147
without attractant PDGF-BB. SMCs on the compact side
were thin, elongated and present only on the surface of the
material in normal culture. The cells on the porous side
were rounded and had migrated into the BC. The definition
of a specific pore size is not relevant in a fibrous hydrogel
as the BC because the nanofibrils can be pushed aside by
migrating cells (Fig. 4B). The density of the nanofibril
network influenced how far SMCs could migrate into the
material. This information is of importance for the design
and manufacture of tubular BC for use as a scaffold for
tissue engineered blood vessels. The luminal side of the
tube would preferably be the smoother and denser compact
side. That might enhance attachment of endothelial cells
since a smoother, dense surface is similar to the basal
membrane. The porous outer side of the tube would
probably promote integration with the host tissue. To
improve cell ingrowth different techniques could be used to
make more free space in the BC pellicle. On way could be
to use leaching techniques, for example introducing
paraffin spheres during culture and then remove them by
leaching. Cellulases are enzymes that degrade cellulose;
these could also be used to alter the density of the BC
pellicle.
To reduce the thrombogenic response in small-diameter
vessels is an important factor to consider. A confluent layer
of endothelial cells on the luminal side of a BC-tube would
probably reduce the risk of thrombus formation. The
behaviour of endothelial cells on the BC surface is
currently being investigated. We think that BC material
with its asymmetric structure and smooth luminal surface
is a good substrate for endothelial cells to form a confluent
layer.
In the stress/strain diagram of a blood vessel, different
components contribute to different parts of the diagram.
The elastin fibrils are involved at the beginning of
stretching while collagen fibrils are responsible for giving
strength at higher elongation and are important closer to
break. The blood vessel is also composed of an extra-
cellular matrix that contributes to load transfer and viscous
behaviour. A pellicle of BC containing 99% water is not as
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2148 H. Bäckdahl et al. / Biomaterials 27 (2006) 2141–2149
strong or elastic as an entire blood vessel that contains
about 70–80% water. However, if BC is to mimic the
collagen fibril network in a blood vessel, it must have a
higher strain at break. These changes can be achieved by
making the pellicle thicker or denser, but cells could also
strengthen the cellulose construct if implanted. The BC
pellicle is composed of 99% water, and we have shown that
cells can migrate into the material. The deformation
behaviour of BC is similar to the porcine carotid artery
(PCA) while the ePTFE shows a totally different deforma-
tion behaviour. While this makes BC promising for
mimicking the mechanical behaviour of a porcine carotid
artery (PCA), the difference in strain at break and
elongation before strain-induced strength is still great. It
must be investigated whether a similarity in elongation is
needed. A tensile test does not give a complete biomecha-
nical picture of the blood vessel since it only gives
information about forces acting in one direction. A better
understanding of the mechanical properties can be
achieved with burst pressure tests and compliance mea-
surements, and this requires a tube of BC. BC grows on the
interface between its culture media and air. So if the air
interface is formed into a tube, BC will produce a tube
around it. We have started to investigate BC-tube
production in another study [34]. Furthermore, the
surrounding environment plays a crucial role in determin-
ing biomechanics in vitro, especially when cells are
involved.
Degradation of BC has not been fully evaluated in either
an in vitro or in vivo setting. Other cellulose-based
materials have however shown limited degradation [35].
The idea of a completely degradable material for tissue
engineering applications is very attractive; in reality,
however, problems with degradable materials are clearly
present but are often neglected. Widely used polymers such
as PGA and PLA yield degradation products that both
chemically and mechanically inhibit cell proliferation in
vitro and induce inflammatory reactions in vivo [36].
Transportation of waste products from resorbed polymers
is also of concern. Degradation of the material by the cells
would be desirable. It is difficult to optimize and
synchronize the degradation time and mechanical proper-
ties of polymers. Keeping the difficulties of degradable
scaffold materials in mind, a non-degradable, biocompa-
tible scaffold that supports cell adhesion, proliferation and
migration is an attractive candidate for future evaluation.
To form a functional blood vessel more cell types than
the SMC must be considered. There are basically three
different cell types in a blood vessel:
Endothelial cells that form the intima, SMC in the media
and fibroblasts in the adventitia. We will perform co-
cultures of booth SMC and EC then we have constructed
the BC into a tubular shape. Our group have previously
shown that cell responses differ greatly between co-cultures
and single culture [37,38].
BC fulfilled several criteria for an optimal scaffold such
as support for attachment, proliferation and cell migration.
The morphology of single nanofibrils, the network
structures of BC and the stress-strain response showed
similarities to that of a collagen network. This opens
opportunities for novel biomimetic scaffold design where
BC replaces the collagen network of the extracellular
matrix.
Thus, BC exhibit properties that are promising for use in
future tissue engineered blood vessels.
Acknowledgements
This work was funded by the Swedish Research Council
(VR 0660), Heart and Lung Foundation, and Vinnova
(2001-06118).
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