An Improved Cannulae
Method
for Bonding
Heparin
to Intravascular
A. J. FREEMAN,C. J, GARDNER, AND M. C. DODDS
The patency of chronically implanted intravascular cannulae is usually limited by thrombus formation at the cannula tip. In the present experiments, methods of improving the antithrombotic properties of the cannulae have been examined using a number of different heparin-bonding treatments. These treatments were evaluated by determining cannula patency in the thoracic aorta of a chronically cannulated rat preparation. Cannulae treated with a heparin-bonding procedure remained patent longer than untreated cannulae. Cannulae treated with tridodecylmethylammonium chloride-heparin-glutaraldehyde remained patent longest (mean patency, 13.3 days compared with 4.0 days in rats with untreated cannulae). This technique provides a simple and effective method of improving the antithrombotic properties of intravascular cannulae by bonding heparin to the lumenal and extralumenal surfaces. Antithrombotic; Key Words: methylammonium chloride
Cannulae;
Heparin-bonding;
Patency; Tridodecyl
INTRODUCTION A common problem encountered in animals with chronically implanted intravascular cannulae is the formation of an occlusive thrombus, usually at the cannula tip, which blocks the cannula and renders the preparation unusable. In an attempt to improve the antithrombotic properties of intravascular cannulae, we have customarily treated them before implantation by sequentially flushing the cannulae with silicone fluid, the quaternary ammonium compound benzalkonium chloride, and heparin. However, we used the method without ever having obtained formal proof that it was effective. Other methods of bonding heparin to plastic cannula materials have been reported in the literature. For example, Grode et al. (1969) tightly bonded large amounts of heparin to polyethylene cannulae using tridodecylmethylammonium chloride (TDMAC). In addition, Lagergren and Eriksson (1971) showed that if glutaraldehyde was used in the final stage of their heparin-bonding procedure to cross-link the attached heparin molecules, cannula patency in a femoral arteriovenous shunt in the dog was increased from 4 hours (with heparin treatment alone) to greater than 12 hours.
From the Cardiovascular sex, United Address Research, Received
Pharmacology
Department,
Glaxo Group
Research,
Ltd.,
Greenford,
Middle-
Kingdom. reprint Ltd.,
requests
to: Dr. M. G. Dodds,
Greenford,
March
1989;
Middlesex revised
Cardiovascular
Pharmacology
Department,
Glaxo Group
UB6 OHE, UK.
and accepted May 1989.
7 Journal of Pharmacological 0 1990 Elsevier
Science
Methods Publishing
23, 7-11 Co.,
Inc.,
0160.5402/90/$3.50
(1990) 655 Avenue
of the Americas,
New
York,
NY 10010
8
A. J. Freeman et al. In the present to see whether preparation.
investigation
we have examined
three heparin-bonding
cannula patency could be improved
in a chronically
treatments
cannulated
rat
METHOD
Cannulae
Preparation
The cannulae were prepared using two sizes of surgical-grade
polyethylene
tubing
(Portex, Ltd.): 30-mm lengths of PP25 tubing (interior diameter, 0.4 mm; exterior diameter, 0.8 mm) heat-sealed to 90-mm lengths of PP50 tubing (interior diameter, 0.58 mm;
exterior
diameter,
0.96 mm).
with the bend as near as possible
Each cannula was formed
to the junction
placing the cannula in the desired it quickly into cold water.
formation
into a U-shape,
of the PP25 and PP50 tubing,
by
into hot water and then transferring
Cannula Treatments Four cannula treatments Treatment The fluid
1 (Silicone
cannulae (MS550,
lution
BDH
USCI,
Ltd.),
Ltd.)
in 0.9%
chloride-heparin)
and then
Chemicals,
5000 units/ml
Treatment
fluid-benzalkonium
were flushed
(Detergicide,
Medical,
were compared:
soaked
then with
and finally
w/v sodium
with
for 10 minutes, a 44%
first
benzalkonium
a sterile
heparin
with
silicone
chloride solution
chloride).
2 (TDMAC-heparin)
A 2%TDMAC-heparin
solution
(as supplied
by Polysciences,
Ltd.) was used after
flushing through a 0.22 pm polypropylene filter. The cannulae were flushed then soaked in TDMAC-heparin solution maintained at 50°C for 2 hours. The nulae were dried minutes.
The
Treatment This drying,
by blowing
a stream
of dry nitrogen
through
cannulae were then left to air dry for several
the lumen
and can-
for 20
hours.
3 (TDMAC-heparin-glutaraldehyde)
treatment
was performed
as described
the cannulae were flushed
for 10 minutes Treatment
so-
(Evans
for TDMAC-heparin,
except that after
and then soaked in a 1% glutaraldehyde
solution
at 60°C. The cannulae were then dried again as before. 4 (Control)
These cannulae were not treated with any heparin-bonding procedure. All the cannulae were sterilized after treatment by total immersion in a dilute
so-
lution of benzalkonium chloride (Detergicide, USCI, Ltd.). Immediately before implantation into the animal, each cannula was flushed, rinsed, and left filled with sterile
0.9%
w/v sodium
Cannula Implantation Adult
chloride
injection
BP (Steriflex).
and Evaluation
male Sprague-Dawley
rats (310-432
g) were used for the evaluation
of can-
nula patency. Cannulae were implanted under general anesthesia induced by pentobarbitone sodium, 50 mg/kg i.p. The left or right carotid artery was exposed
9
16
12
*
10
T
8
6
i
Treatment 1 = Silicone fluid-benzalkonium taraldehyde; 4 = Control.
chloride-heparin;
2 = TDMAC-heparin;
3 = TDMAC-heparin-glu-
Values are means +- SEM; n = 7. Significant difference
FIGURE 1.
from control (4):‘~
< 0.05 **p < 0.01 (Student’s t test).
The effects of four heparin~bonding
treatments on cannula patency in viva.
10
A. j. Freeman et al. through
a ventral
the midcervical
midline
region.
incision
A blunt
in the neck distal to the pharynx
(nonbevelled)
cannula
aortic arch by inserting the cannula into the carotid artery proximal and passing it toward the heart for 30 mm so that the PP25 portion lay within
the vessel,
to that described
with the tip in the thoracic
by Popovic
and Popovic
heparinized
saline (250 units/ml
steel plugs,
and the animals
The patency considered
of each implanted
to be patent
aorta.
if blood
chloride),
were
in
into the
to the ligation of the cannula
This procedure
(1960). The cannulae
in 0.9% w/v sodium
allowed
and ligated
was then implanted
was similar
left filled with
sealed with stainless-
to recover.
cannula came
was determined back freely
every day. A cannula
into the cannula
when
was
the ex-
teriorized end was exposed to atmospheric pressure. The cannula was then flushed and left filled with a sterile heparin solution (250 units/ml in 0.9% w/v sodium chloride).
RESULTS Cannulae untreated
treated
with a heparin-bonding
cannulae
(Figure 1). Implanted
ment 4) had a mean patency of cannulae
treated
with
1) or TDMAC-heparin treated
cannulae,
tistically
silicone
(treatment although
significant
Treatment
duration
(treatment
procedure cannulae
remained
patent
longer than
that had not been treated
of only 4.0 ? 1.4 days. The patency
fluid-benzalkonium 2) was increased
chloride-heparin twofold
in the case of treatment 1, p = 0.032;
(treatment
in comparison
2 the difference
with
un-
was not sta-
2, p = 0.11, Student’s
treatment
(treat-
duration
t test).
of cannulae with TDMAC-heparin-glutaraldehyde (treatment 3) produced (3.3-fold) Increase in patency duration (13.3 * 2.0 days compared with 4.0
a greater ? 1.4 days for the untreated of cannulae bonded in comparison with 0.065 and 0.091,
cannula
group, p < 0.01).However
the patency duration
with glutaraldehyde (treatment 3) was not significantly greater the other heparin-bonding methods (treatments 1 and 2, p =
respectively).
DISCUSSION The results of this investigation the period
of cannula
patency,
suggest that heparin-bonding probably
thrombogenic.
The greatest
improvement
nulae occurred
in the group of rats in which
by rendering in patency
treatments
the cannula duration
extend
material
of implanted
TDMAC-heparin-glutaraldehyde
less can(treat-
ment 3) was used to bond heparin to the polyethylene cannulae. The glutaraldehyde treatment, by cross-linking the attached heparin molecules, presumably resulted in a more stable heparin coating than that produced by the TDMAC-heparin alone (treatment 2). The procedure appears to provide a simple and effective method of improving the antithrombotic properties of polyethylene intravascular cannulae. Since the TDMAC-heparin technique has been reported to bond heparin to a including silicone rubber, Teflon, and polyvinylchloride variety of substrates, (Grode
et al., 1969), incorporation
of glutaraldehyde
in cannula
treatment
may also
Heparin Bonding to Intravascular Cannulae improve
the antithrombotic
terials other The authors throughout
properties
of intravascular
cannulae
made
from
ma-
than polyethylene. wish to thank Mrs.
this
C. A. Ransom
and Miss
M. Fergus
for their
expert
technical
assistance
investigation.
REFERENCES Grode
GA, Anderson
JJ, Grotta
(1969) Nonthrombogenic coating
process.
Trans
HM,
materials Am Sot
Attif
and Falb RP
arin:
via a simple
Artif
Intern
Or-
gans 15:1-6. Lagergren
Popovic nulation
HR and Eriksson
a stable surface
Preparation Intern
monolayer
JC (1971) Plastics
with
of cross-linked
hep-
ground
Organs
and evaluation.
V and Popovic of
aorta
squirrels.
Trans
Am Sot
17:10-12. and
P (1960) vena
Permanent cava in
/ Appl Physiol
rats
15~727-728.
canand
11
Method
for Bonding
Heparin
to Intravascular
A. J. FREEMAN,C. J, GARDNER, AND M. C. DODDS
The patency of chronically implanted intravascular cannulae is usually limited by thrombus formation at the cannula tip. In the present experiments, methods of improving the antithrombotic properties of the cannulae have been examined using a number of different heparin-bonding treatments. These treatments were evaluated by determining cannula patency in the thoracic aorta of a chronically cannulated rat preparation. Cannulae treated with a heparin-bonding procedure remained patent longer than untreated cannulae. Cannulae treated with tridodecylmethylammonium chloride-heparin-glutaraldehyde remained patent longest (mean patency, 13.3 days compared with 4.0 days in rats with untreated cannulae). This technique provides a simple and effective method of improving the antithrombotic properties of intravascular cannulae by bonding heparin to the lumenal and extralumenal surfaces. Antithrombotic; Key Words: methylammonium chloride
Cannulae;
Heparin-bonding;
Patency; Tridodecyl
INTRODUCTION A common problem encountered in animals with chronically implanted intravascular cannulae is the formation of an occlusive thrombus, usually at the cannula tip, which blocks the cannula and renders the preparation unusable. In an attempt to improve the antithrombotic properties of intravascular cannulae, we have customarily treated them before implantation by sequentially flushing the cannulae with silicone fluid, the quaternary ammonium compound benzalkonium chloride, and heparin. However, we used the method without ever having obtained formal proof that it was effective. Other methods of bonding heparin to plastic cannula materials have been reported in the literature. For example, Grode et al. (1969) tightly bonded large amounts of heparin to polyethylene cannulae using tridodecylmethylammonium chloride (TDMAC). In addition, Lagergren and Eriksson (1971) showed that if glutaraldehyde was used in the final stage of their heparin-bonding procedure to cross-link the attached heparin molecules, cannula patency in a femoral arteriovenous shunt in the dog was increased from 4 hours (with heparin treatment alone) to greater than 12 hours.
From the Cardiovascular sex, United Address Research, Received
Pharmacology
Department,
Glaxo Group
Research,
Ltd.,
Greenford,
Middle-
Kingdom. reprint Ltd.,
requests
to: Dr. M. G. Dodds,
Greenford,
March
1989;
Middlesex revised
Cardiovascular
Pharmacology
Department,
Glaxo Group
UB6 OHE, UK.
and accepted May 1989.
7 Journal of Pharmacological 0 1990 Elsevier
Science
Methods Publishing
23, 7-11 Co.,
Inc.,
0160.5402/90/$3.50
(1990) 655 Avenue
of the Americas,
New
York,
NY 10010
8
A. J. Freeman et al. In the present to see whether preparation.
investigation
we have examined
three heparin-bonding
cannula patency could be improved
in a chronically
treatments
cannulated
rat
METHOD
Cannulae
Preparation
The cannulae were prepared using two sizes of surgical-grade
polyethylene
tubing
(Portex, Ltd.): 30-mm lengths of PP25 tubing (interior diameter, 0.4 mm; exterior diameter, 0.8 mm) heat-sealed to 90-mm lengths of PP50 tubing (interior diameter, 0.58 mm;
exterior
diameter,
0.96 mm).
with the bend as near as possible
Each cannula was formed
to the junction
placing the cannula in the desired it quickly into cold water.
formation
into a U-shape,
of the PP25 and PP50 tubing,
by
into hot water and then transferring
Cannula Treatments Four cannula treatments Treatment The fluid
1 (Silicone
cannulae (MS550,
lution
BDH
USCI,
Ltd.),
Ltd.)
in 0.9%
chloride-heparin)
and then
Chemicals,
5000 units/ml
Treatment
fluid-benzalkonium
were flushed
(Detergicide,
Medical,
were compared:
soaked
then with
and finally
w/v sodium
with
for 10 minutes, a 44%
first
benzalkonium
a sterile
heparin
with
silicone
chloride solution
chloride).
2 (TDMAC-heparin)
A 2%TDMAC-heparin
solution
(as supplied
by Polysciences,
Ltd.) was used after
flushing through a 0.22 pm polypropylene filter. The cannulae were flushed then soaked in TDMAC-heparin solution maintained at 50°C for 2 hours. The nulae were dried minutes.
The
Treatment This drying,
by blowing
a stream
of dry nitrogen
through
cannulae were then left to air dry for several
the lumen
and can-
for 20
hours.
3 (TDMAC-heparin-glutaraldehyde)
treatment
was performed
as described
the cannulae were flushed
for 10 minutes Treatment
so-
(Evans
for TDMAC-heparin,
except that after
and then soaked in a 1% glutaraldehyde
solution
at 60°C. The cannulae were then dried again as before. 4 (Control)
These cannulae were not treated with any heparin-bonding procedure. All the cannulae were sterilized after treatment by total immersion in a dilute
so-
lution of benzalkonium chloride (Detergicide, USCI, Ltd.). Immediately before implantation into the animal, each cannula was flushed, rinsed, and left filled with sterile
0.9%
w/v sodium
Cannula Implantation Adult
chloride
injection
BP (Steriflex).
and Evaluation
male Sprague-Dawley
rats (310-432
g) were used for the evaluation
of can-
nula patency. Cannulae were implanted under general anesthesia induced by pentobarbitone sodium, 50 mg/kg i.p. The left or right carotid artery was exposed
9
16
12
*
10
T
8
6
i
Treatment 1 = Silicone fluid-benzalkonium taraldehyde; 4 = Control.
chloride-heparin;
2 = TDMAC-heparin;
3 = TDMAC-heparin-glu-
Values are means +- SEM; n = 7. Significant difference
FIGURE 1.
from control (4):‘~
< 0.05 **p < 0.01 (Student’s t test).
The effects of four heparin~bonding
treatments on cannula patency in viva.
10
A. j. Freeman et al. through
a ventral
the midcervical
midline
region.
incision
A blunt
in the neck distal to the pharynx
(nonbevelled)
cannula
aortic arch by inserting the cannula into the carotid artery proximal and passing it toward the heart for 30 mm so that the PP25 portion lay within
the vessel,
to that described
with the tip in the thoracic
by Popovic
and Popovic
heparinized
saline (250 units/ml
steel plugs,
and the animals
The patency considered
of each implanted
to be patent
aorta.
if blood
chloride),
were
in
into the
to the ligation of the cannula
This procedure
(1960). The cannulae
in 0.9% w/v sodium
allowed
and ligated
was then implanted
was similar
left filled with
sealed with stainless-
to recover.
cannula came
was determined back freely
every day. A cannula
into the cannula
when
was
the ex-
teriorized end was exposed to atmospheric pressure. The cannula was then flushed and left filled with a sterile heparin solution (250 units/ml in 0.9% w/v sodium chloride).
RESULTS Cannulae untreated
treated
with a heparin-bonding
cannulae
(Figure 1). Implanted
ment 4) had a mean patency of cannulae
treated
with
1) or TDMAC-heparin treated
cannulae,
tistically
silicone
(treatment although
significant
Treatment
duration
(treatment
procedure cannulae
remained
patent
longer than
that had not been treated
of only 4.0 ? 1.4 days. The patency
fluid-benzalkonium 2) was increased
chloride-heparin twofold
in the case of treatment 1, p = 0.032;
(treatment
in comparison
2 the difference
with
un-
was not sta-
2, p = 0.11, Student’s
treatment
(treat-
duration
t test).
of cannulae with TDMAC-heparin-glutaraldehyde (treatment 3) produced (3.3-fold) Increase in patency duration (13.3 * 2.0 days compared with 4.0
a greater ? 1.4 days for the untreated of cannulae bonded in comparison with 0.065 and 0.091,
cannula
group, p < 0.01).However
the patency duration
with glutaraldehyde (treatment 3) was not significantly greater the other heparin-bonding methods (treatments 1 and 2, p =
respectively).
DISCUSSION The results of this investigation the period
of cannula
patency,
suggest that heparin-bonding probably
thrombogenic.
The greatest
improvement
nulae occurred
in the group of rats in which
by rendering in patency
treatments
the cannula duration
extend
material
of implanted
TDMAC-heparin-glutaraldehyde
less can(treat-
ment 3) was used to bond heparin to the polyethylene cannulae. The glutaraldehyde treatment, by cross-linking the attached heparin molecules, presumably resulted in a more stable heparin coating than that produced by the TDMAC-heparin alone (treatment 2). The procedure appears to provide a simple and effective method of improving the antithrombotic properties of polyethylene intravascular cannulae. Since the TDMAC-heparin technique has been reported to bond heparin to a including silicone rubber, Teflon, and polyvinylchloride variety of substrates, (Grode
et al., 1969), incorporation
of glutaraldehyde
in cannula
treatment
may also
Heparin Bonding to Intravascular Cannulae improve
the antithrombotic
terials other The authors throughout
properties
of intravascular
cannulae
made
from
ma-
than polyethylene. wish to thank Mrs.
this
C. A. Ransom
and Miss
M. Fergus
for their
expert
technical
assistance
investigation.
REFERENCES Grode
GA, Anderson
JJ, Grotta
(1969) Nonthrombogenic coating
process.
Trans
HM,
materials Am Sot
Attif
and Falb RP
arin:
via a simple
Artif
Intern
Or-
gans 15:1-6. Lagergren
Popovic nulation
HR and Eriksson
a stable surface
Preparation Intern
monolayer
JC (1971) Plastics
with
of cross-linked
hep-
ground
Organs
and evaluation.
V and Popovic of
aorta
squirrels.
Trans
Am Sot
17:10-12. and
P (1960) vena
Permanent cava in
/ Appl Physiol
rats
15~727-728.
canand
11
