AbstractObjective: to investigate in a pig model whether small diameter ePTFE grafts will sustain a confluent endothelial cell layer formed in vitro under shear stress conditions. Material and Methods: thirteen ePTFE (4 mm) grafts were implanted end to end in the right femoral artery of; 8 grafts had been endothelialized in vitro. Grafts were left in situ for 6 weeks then evaluated with ultrasound and histology. Results: seven endothelialized graft were patent with confluent endothelial cell lining. None of the control grafts were patent or showed evidence of an endothelial lining. Conclusion: in this pig model ePTFE grafts sustained for 6 weeks a confluent endothelial cell layer formed in vitro under shear stress.Eur J Vasc Endovasc Surg 26, 156-160 (2003

Büttemeyer, R.

Mall, J.W.

Paulitschke, M.

Philipp, A.W.

Rademacher, A.

Elsevier - Publisher Connector 

In a Pig Model ePTFE Grafts will Sustain For 6 Weeks a Confluent
Endothelial Cell Layer Formed In Vitro Under Shear Stress Conditions
R. BuÈ ttemeyer1, J. W. Mall1, M. Paulitschke2, A. Rademacher2 and A. W. Philipp2
1Department of General, Visceral, Thoracic and Vascular Surgery, Medical Faculty of the Humboldt University,
and 2Cell-Lining GmbH, Gesellschaft fuÈr Zellkulturen, ChariteÂ, Campus Mitte, Schumannstr. 20/21,
10117 Berlin, Germany
Objective: to investigate in a pig model whether small diameter ePTFE grafts will sustain a confluent endothelial cell layer
formed in vitro under shear stress conditions.
Material and Methods: thirteen ePTFE (4 mm) grafts were implanted end to end in the right femoral artery of;
8 grafts had been endothelialized in vitro. Grafts were left in situ for 6 weeks then evaluated with ultrasound and
histology.
Results: seven endothelialized graft were patent with confluent endothelial cell lining. None of the control grafts were
patent or showed evidence of an endothelial lining.
Conclusion: in this pig model ePTFE grafts sustained for 6 weeks a confluent endothelial cell layer formed in vitro under
shear stress.
Key Words: Tissue engineering; Vascular grafts; Shear stress.
Introduction
Autologous saphenous vein is the preferred conduit
for femoro-distal bypass but is often unavailable.1 The
patency rates of small diameter ePTFE grafts are dis-
appointing2±10 partly because of the absence of an endo-
thelial cell lining.11±16 However, the artificial seeding
of ePTFE grafts with autologous harvested endothelial
cells failed to improve patency in vivo,17±19 possibly
because such cells fail to adhere on to the graft under
normal blood flow conditions.20,21 We have previously
reported the endothelialization of ePTFE grafts under
shear stress22 and were able to demonstrate that such
grafts sustained endothelial cell confluency when
exposed to pulsed physiological flow conditions.23
Therefore, with regard to the potential use of flow
processed ePTFE grafts as a substitute to autologous
bypass, a prospective animal study was carried out.
The aim of this study was to extend these observations
to a pig model.
Materials and Methods
Thirteen 25±30 kg, 10±14 week female pigs (Deutsche
Landrasse, Charles River Wiga, Ansbach, Germany)
were housed under standard laboratory conditions,
allowed food and water ad libitum and randomized
into two groups. Group 1 (n 5) received an uncoated
4 mm ePTFE graft and group 2 (n 8) a shear stress
endothelialized 4 mm ePTFE graft.
For endothelial cell harvest and cultivation the ani-
mals were anaesthetized with i.m. Stresnil (Azapiron,
Janssen, Germany) 4 mg/kg, 0.05 mg Atropine and
Ethomidat-Lipiro 0.15±0.3 mg/kg. The external jugular
vein was dissected over 5 cm, repeatedly flushed with
Dulbecco's PBS (Biochrom AG, Berlin, Germany), filled
with 1 U/ml Collagenase P1 (Boehringer, Mannheim,
Germany) diluted in PBS and incubated at 37 C (gazed
5% CO2 at 100% humidity) for 15 min. Collagenase
activity was stopped with 10% FCS supplemented
standard RPMI medium (Biochrom AG, Berlin,
Germany). After centrifugation at 1000 rpm for 5 min
thesupernatantwasrejectedandthepellet resuspended
in PBS. This procedure was repeated twice before
the pellet was suspended in Dulbecco's modified
Eagle's medium (DMEM) (Sigma±Aldrich, Steinheim,
Germany) and Endothelial Cell Growth Medium
(ECGM) (PromoCell GmbH, Germany), mixed 1:1.
Please address all correspondence to: R. BuÈ ttemeyer, Department
of General, Visceral, Thoracic and Vascular Surgery, Medical Faculty
of the Humboldt University ChariteÂ, Campus Mitte, Schumannstr.
20/21, 10117 Berlin, Germany.
Eur J Vasc Endovasc Surg 26, 156±160 (2003)
doi:10.1053/ejvs.2002.1881, available online at http://www.sciencedirect.com on
1078±5884/03/020156 05 $35.00/0 # 2003 Elsevier Science Ltd. All rights reserved.
Cells were cultivated in an incubator at (37 C, 5% CO2
at 100% humidity) and grown to subconfluence (80%
confluent) before passaging. Harvesting was per-
formed by incubating the cell monolayer 5 min with
PBS diluted Trypsin/EDTA solution (Biochrom AG,
Germany). Trypsin was inactivated by adding 10%
FCS supplemented standard RPMI medium. After cen-
trifugation and washing in PBS the cells were seeded
again at a ratio of 1:2 to 1:4 until final harvest. Cell
number and viability were controlled with an electron-
ic cell counting system. Cell identity was confirmed in
situ with von Willebrand/Factor VIII staining (Sigma±
Aldrich, Steinheim, Germany) using the EnVisionTM
AP detection system (Dako, Hamburg, Germany) and
by observing the typical cobblestone morphology of
endothelial cells in confluent monolayer. Contamin-
ation by Mycoplasma and other bacteria was tested
routinely by a fluorescence DNA staining kit
(Biochrom AG, Berlin, Germany). Photographic docu-
mentation of cell growth was performed under light
microscopy daily and before new passages of the cells.
Control cultures of endothelial cells were performed
for each harvest to check growth characteristics and cell
viability. Data were tested for normal distribution by
Spearman's correlation test. Categorical data are dis-
played as mean and range.
The ePTFE graft (Model: T04030C30, Baxter,
Vascular Systems Division, Illinois, U.S.A.) was placed
in the sterile perfusion chamber TCS2c (Cell-Lining
GmbH, Berlin, Germany), which was configured
with two separate, an inner and outer, circulation.
The inner circulation was adjustable to different graft
extents. The outer perfusion circuit was run as static
(no pump connected, medium reservoirs were on the
same level as perfusion chamber and outer circula-
tion). Inner and outer perfusion circuit's medium
reservoirs were fitted with a sterile membrane filter,
guaranteeing gas exchange between medium and well
defined atmosphere within the incubator, in order to
keep the pressure inside the chamber constant. For
further chamber specifications see Figure 1. For initial
fibrin coating the graft was filled air bubble free with
Beriplast1 HS fibrin glue (Aventis Behring, Lieder-
bach, Germany). The inlet was then pressurized
using a simple 20 ml syringe until small fibrinogen
drops could be observed on the graft's outside, indi-
cating total `` sweating'' of the porous material hence
displacing the air. Surplus fibrinogen was removed by
blowing the graft with air. It was then filled up with
diluted thrombin solution, air bubble free, which was
immediately removed again by blow out. This proced-
ure was repeated until the blown-out thrombin stayed
liquid. The inner perfusion circuit was then filled with
the prepared endothelial cell suspension and, after a
seeding period of 4 h immediately connected to a peri-
staltic pump. The perfusion experiments were all
conducted with Dulbecco's modified Eagle's medium
(DMEM) supplemented with: 25 mg Amphotericin B
(0.05 mg/ml), 5 ml Heparin (1000 U/ml) diluted in
cell-culture tested water (50 U/ml) 50 ml FCS (10%),
10 ml l-Glutamin 200 mM (4 mM), 10 ml Hepes 1 M
(0.02 M), 5 ml Pen/Strep (100 U/ml) (Sigma±Aldrich,
Steinheim, Germany) containing 10% fetal calf serum
and ECGM. This was supplemented with: ECGS/H
(0.4%), FCS (2%), Epidermal Growth Factor 0.1 ng/ml,
Hydrocortison (1 mg/ml), Basic Fibroblast Factor
(1 ng/ml), Amphotericin B (50 ng/ml), Gentamicin
(50 mg/ml)) (PromoCell GmbH, Germany), mixed 1:1.
A constant flow was applied and slowly increased
over the first 24 h with a maximum flow rate of
12.25 ml/s corresponding to a maximum shear stress
rate of 15 dyn/cm2. After 24 h the flow was pulsed
with shear rate peaks of 15 dyn/cm2 to simulate
human blood flow circumstances. Shear rate values
were calculated with the Hagen±Poiseuille equation:
t 4Z Q/(p  r3). Viscosity of DMEM was deter-
mined at 37 C with Low-Shear-Viscosimeter LS 40
(Mettler Toledo, Zurich, Switzerland). The time point
of endothelial cell confluence was determined via
the established method based on the Bernouilli
effect.23
Fourteen days later, endothelialized ePTFE grafts
were implanted end-to-end into the right femoral
artery under general anaesthesia. A 7 cm right groin
incision was performed and the right femoral artery
dissected over 6 cm distal to the inguinal ligament.
The flow at the artery was determined by Doppler
ultrasound (Transonic Systems Inc., Ithaca, U.S.A.).
The deep femoral artery was ligated to prevent early
collateralization. The superficial femoral artery was
Outer Perfusion
Circuit 
Inner Perfusion
Circuit
(ePTFE-Prosthesis)
Adjustable Lids
Fig. 1. Perfusion Chamber TCS2c.
ePTFE Grafts in a Pig Model 157
Eur J Vasc Endovasc Surg Vol 26, August 2003
then clamped and incised over 4 mm and flushed
distally and proximally direction heparin (2500 IU
heparin in 250 ml saline). No systemic heparin or
anti-fibrinolytic agents were administered. A 5 cm
length of the superficial femoral artery was excised
just below the inguinal ligament. A non-endothelia-
lized (n 5) or an under shear stress endothelialized
(n 8) 4 mm ePTFE graft was interposed in an end-to-
end fashion. The arterial blood flow was remeasured.
The incision was closed by running subcutaneous
stitches and skin stapling.
Temperature, body weight, activity status and
food intake were monitored daily. Eight days after
implantation, graft flow was measured with a hand-
held Doppler ultrasound twice weekly. Six weeks after
implantation animals were re-anaesthetised, the inci-
sion reopened, the bypass was dissected, and arterial
blood flow measured (Transonic1 Flowprobe). The
grafts were excised and placed in 4% formalin for
histology.
Grafts were embedded in paraffin and sectioned at
in the middle and at both end. Haematoxylin and
eosin slides were prodcued in a standard manner.
Endothelial cell verification was performed by
peroxidase±antiperoxidase immunhistochemistry.
Using antibodies against endothelial cell typical clus-
ters of differentiation 31 as well as von Willebrand/
Factor VIII.
Results
Endothelial cell harvest and cultivation was con-
ducted without complications or contamination.
Overall mean cell gain was 1 590 475 (range:
1123 800±3 360 000) cells/ml per vein after a mean
cultivation time of 11.25 (range: 9±13) days. Mean
cell vitality was 92.5% (range: 89±96%). For graft cul-
tivation 75 000 cells/cm2 graft lumen were seeded.
After the initial roller period of 4 h, a cell number of
71±96% was calculated as being adherent to the grafts
surface. Histologically, this corresponds to a conflu-
ence of 35±50% on the porous surface depending on
the varying cell diameter of the endothelial cells used
in our experiments.22 After 4 h, perfusion of the inner
circuit was started and steadily increased to a shear
stress rate of 15 dyn/cm2 (12.25 ml/s) after 24 h. The
perfusion scheme was then altered to pulsation, with
shear stress peaks of 15 dyn/cm2 (Fig. 2). Over the
whole perfusion period the perfusion chamber weight
was closely monitored four times an hour in order to
determine cell confluency based on the established
method following the Bernouilli principle. Mean
perfusion time for each graft was 86.5 h (range:
65.5±161.5 h). No signs of infection or bacterial
0 4 8 12 16 20 24 28 32
Time (h)
Sh
ea
r 
ra
te
 (
dy
n/
cm
²)
0
2
4
6
8
10
12
14
16
Fig. 2. Shear stress rates under perfusion culture.
Fig. 3. Perfusion rates directly after implantation vs before explantation.
158 R. BuÈ ttemeyer et al.
Eur J Vasc Endovasc Surg Vol 26, August 2003
0
50
100
150
200
250
300
60 20 90 92 94 18 15 58 65 91 93 95
Directly after implantation Before explantation (Endothelialized)
Animal number
Pe
rf
us
io
n 
ra
te
 (m
l/m
in)
contamination were seen throughout the repeated
testing in the whole series and in control cultures.
As one pig was lost after implantation from malig-
nant hyperthermia, only 12 grafts were evaluable.
Otherwise, surgery and follow up were uneventful.
In the endothelialized group limb development was
normal (average diameter 51 cm) and there was no
loss of function. In the control group one hind limb
was underdeveloped (diameter 41 cm) in association
with a graft infection. The other 11 prosthetic grafts
were surrounded by typical fibrous tissue. At 6 weeks
all but one of the endothelialized grafts showed nor-
mal pulsation in contrast to the control grafts which
were all non-pulsatile.
Mean flow directly after implantation of the pro-
cessed graft group was 187 (range: 182±193) ml/min,
and prior to explantation was 225 (range: 220±250)
ml/min (Fig. 3). No perfusion could be measured in
the grafts implanted not endothelialized. Flow was
demonstrated in all but one endothelialized graft up
to explantation time by ultrasound control. In the
unprocessed grafts, flow could only be verified up to
the beginning of the fourth week after implantation.
An endothelial cell lining was found in seven
endothelialized grafts removed from living animals
at 6 weeks (Figs 4 and 5). Only one graft showed
thrombotic occlusion and endothelial cell detection
was weak. The endothelialized graft in the animal
deceased of malignant hyperthermia also demon-
strated a confluent endothelial cell lining. In control
grafts no endothelial cell-lining could be verified. All
grafts were partly or completely filled with either
organized thrombotic material and/or granulation
and collagen rich scar tissue (Fig. 6).
Discussion
We have previously established in vitro that endo-
thelial cells grown under shear stress conditions do
not only show a higher proliferation rate, but also
better ePFTE anchorage.22±25 We chose a pig model
to extend these observations because pigs, like
humans, do not spontaneously form an endothelial
monolayer.26 After 6 weeks in vivo all but one endothe-
lialized graft showed good blood flow. By compari-
son, all unprocessed grafts were occluded. Histology
of the endothelialized grafts verified an endothelial
cell layer integrity. Further studies are required to
optimize the seeding process, to determine the long
term durability of the endothelial layer in vivo, and to
adapt the process for use in patients.
Surrounding tissue 
ePTFE-Prosthesis
von Willebrand
(Faktor VIII) stained
Endothelium
Fig. 5. Endothelialized graft after 6 weeks in vivo, VWF (factor VIII)
staining.
Surrounding tissue 
ePTFE-Prosthesis
Endothel
Fig. 4. Endothelialized graft after 6 weeks in vivo, CD 31 staining.
Collagen rich granulation- and scar tissue
ePTFE-Prosthesis
Fig. 6. Non-endothelialized graft after 6 weeks in vivo, Animal 60,
HE staining.
ePTFE Grafts in a Pig Model 159
Eur J Vasc Endovasc Surg Vol 26, August 2003
Acknowledgments
This work was supported by Cell-Lining GmbH (Berlin, Germany).
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Accepted 13 January 2003
160 R. BuÈ ttemeyer et al.
Eur J Vasc Endovasc Surg Vol 26, August 2003


In a pig model ePTFE grafts will sustain for 6 weeks a confluent endothelial cell layer formed in vitro under shear stress conditions 

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In a pig model ePTFE grafts will sustain for 6 weeks a confluent endothelial cell layer formed in vitro under shear stress conditions

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