Journal of Investigative Surgery, 14:55 61, 2001
Copyright c 2001 Taylor & Francis
0894-1939/01 $12.00 + .00
A Novel Technique in a Sheep Model
for Evaluating Prosthetic Heart
Valve Performance
Matthew M. Puc, MD,
Steven W. Marra, MD,
Hoang S. Tran, MD,
Jonathan H. Cilley, Jr., MD,
Charles W. Hewitt, PhD, and
Anthony J. DelRossi, MD
Department of Surgery, Cooper
Hospital/University Medical
Center, University of Medicine
and Dentistry of New Jersey,
Robert Wood Johnson Medical
School, Camden, New Jersey,
USA
Received 8 October 1999;
accepted 26 June 2000.
Presented at the 15th Annual Meeting
of the Academy of Surgical Research,
2 4 September 1999, Scottsdale, AZ.
Address correspondence to Charles
W. Hewitt, PhD, Cooper Hospital/UMC,
Three Cooper Plaza, Suite 411,
Camden, NJ 08103, USA. E-mail:
chewitt@umdnj.edu
New Methodologies
ABSTRACT There have been many various animal studies to evalu-
ate the structural integrity and antithrombogenicity of prosthetic heart
valves. We were interested in developing a novel sheep model to study
the thrombogenicity of mechanical heart valves placed into the systemic
circulation but without the need for cardiac bypass. Also, we wanted to
minimize the risk of paraplegia from complete thoracic aortic clamping.
Six sheep underwent left lateral thoracotomy for placement of a mechan-
ical heart valve in parallel with the descending thoracic aorta. A valved
conduit with a dacron tube graft sutured to the back end was fashioned.
Employing partial aortic occlusion with a side-biting clamp, the proximal
and distal ends were anastomosed in an end-to-side fashion. Once ow
was con rmed through the graft, the native aorta was occulded with um-
bilical tape. The sheep received no postoperative anticoagulation. The
median operative time and estimated blood loss (EBL) was 170 min and
250 cc, respectively. Patency of the valved conduits was con rmed during
the initial procedure, and there was no incidence of paraplegia postop-
eratively. Two animals expired shortly after extubation and at necropsy
the valved conduits were patent with preserved valve function. The four
survivors were sacri ced a median of 37 days postoperatively. Prior to
euthanasia, the valved conduits were evaluated in situ with ultrasound.
In all cases, the valves had clot formation at the hinges, which prevented
active movement of the lea ets. This novel in vivo technique provides an
alternative in testing the thrombogenicity of prosthetic heart valves with-
out cardiac bypass or the risk of paraplegia in an animal that is extremely
sensitive to complete aortic cross-clamp.
KEYWORDS heart valve, sheep model, partial occlusion
55
New Methodologies
P
rosthetic heart valves have evolved signi -
cantly since their initial clinical use in cardiac
surgery. Today, more than 60,000 heart valve
replacements are performed in the United States an-
nually [1]. The two main types of heart valves are
either mechanical, which are composed primarily of
metal or carbon alloys, or bioprostheses. These bio-
prostheses are further divided into heterografts, such
as porcine or bovine tissue, or homografts, which are
preserved human aortic valves.
Our area of interest has involved mechanical heart
valves, in particular the bilea et tilting disk (St. Jude
Medical) valve. Even though this type of mechan-
ical valve has the lowest thrombogenic potential,
patients still require lifelong anticoagulation [2]. In-
dividuals taking oral anticoagulation are suscepti-
ble to hemorrhagic complications, particularly in
the gastrointestinal tract, cerebral circulation, and
retroperitonium. The incidence of these complica-
tions has been reported to be approximately 1% to
4% per patient-year [3]. Our efforts in identifying
a solution that would eliminate the need for life-
long anticoagulation have been centered around sur-
face modi cation of mechanical heart valves to ef-
fect thrombogenicity [4]. We have employed two
separate techniques, which are well described in the
literature and are only mentioned brie y here. One
process, called diamond-like carbon coating (DLC),
produces a dense amorphous hydrocarbon polymer
coating [5]. The other process, plasma or glow dis-
charge treatment (GDT), is a stepwise reaction utiliz-
ing gas plasma to generate a new molecular property
on the surface of the mechanical valve [6]. These
modi cations change the surface layers without af-
fecting the bulk structure or durability. Early reports
from our research laboratory employing both types
of surface modi cations have shown promising re-
sults, but the nal interpretation is yet to be deter-
mined [7]. Therefore, we embarked on an in vivo
study to determine the true potential of these two
processes.
We rst had to establish a reliable animal model
that would satisfy our requirements in studying the
surface modi cation of mechanical heart valves. In
choosing the proper animal model, the sheep was the
most appropriate. As the literature supports, sheep
are an excellent animal model, since they are compa-
56
rable to humans in various ways [ 8, 9]. Their blood
pressure, cardiac output, heart rate, and blood coagu-
lation pro le mimic those of humans [10, 11]. Sheep
also have a docile temperament that allows their care
to be relatively simple [ 12]. In addition, anesthetic
doses in sheep are similar to humans, which makes
for easy administration of anesthesia [13, 14].
When designing this model, it was necessary to
make the model simple and ef cient with a minimal
expenditure of resources. One way to accomplish
this would be to avoid cardiac bypass and its associ-
ated risks, but there was a desire to place the valve
within the systemic circulation to mimic as closely
as possible the pressure gradients of an orthotopic
position. This could potentially be achieved by po-
sitioning the valve in the descending thoracic aorta,
as previously reported, but would necessitate cross-
clamping the aorta [ 15, 16] . This last point is criti-
cal since our prior experience has shown that sheep
do not tolerate complete thoracic cross-clamping for
extended periods. This was seen in our laboratory,
where we experienced paralysis following as little
as 16 mins of total aortic cross-clamping. With all
of these factors taken into consideration, the nal
model incorporated a valved conduit anastomosed
in parallel with the descending thoracic aorta that
could be accomplished with a partial cross-clamping
technique.
MATERIALS AND METHODS
All experiments were performed according to a re-
search protocol approved by the University of Medi-
cine and Dentistry of New JerseyRobert Wood
Johnson Medical School (Camden, N J) Institutional
Animal Care and Use Committee (IACUC).
Preoperative Care
The adult sheep were transported from a pasture
farm and housed in a sheep pen in the vivarium facil-
ities. The vivarium staff was instrumental in the care
of the sheep prior to the operative procedure and
postoperatively. All the necessary materials, medica-
tions, and equipment were properly prepared the day
prior to surgery (Tables 1 and 2). Appropriate feed
and water were dispensed, and the sheep were fasted
for approximately 12 h prior to surgery.
M. M. PUC ET AL.

TABLE 1 Medications
Anesthetic
agents Antibiotics Analgesics Euthanasia
Ketamine Ampicillin Meperidine HCL Somelethal
Thiamylalal Gentamycin Buprenorpherine
sodium HCL
Halothane
Operative Procedure
At the time of surgery, anesthesia was induced
using intramuscular (im) injections of ketamine (30
45 mg/kg, Parke-Davis, Morris Plain, NJ) and thi-
amylalal sodium (814 mg/kg). Once adequately se-
dated, the vivarium staff moved the sheep to the op-
erating room and properly positioned the animal for
intubation. After orotracheal intubation, the animal
was maintained on 24% halothane gas (Halocar-
bon Laboratories, River Edge, NJ) volatilized with
oxygen. Tidal volume was set at 10 mL/kg plus ad-
ditional volume for the dead space within the tub-
ing circuit. The respiratory rate was maintained be-
tween 7 and 20 breaths/min. Electrocardiographic
leads were placed to monitor the heart rate and the
cardiac rhythm. An intravenous catheter was inserted
percutaneously into the cephalic vein. Normal
saline (0.9%) was used for maintenance uid at a
rate of 10 mL/kg. A femoral arterial line for contin-
uous arterial blood-pressure monitoring was placed
via a cut-down method. Intravenous doses of gen-
tamycin (2 mg/kg, Elkins-Sinn, Inc., Cherry Hill,
NJ) and ampicillin sodium (7 mg/kg, Bristo-Myers
Squibb Company, Princeton, NJ) were then given.
The animal was placed in the right lateral decubi-
TABLE 2 Materials and equipment
0.9% Sodium chloride
Sterile water
Angiocatheters
iv Sets and tubing
Surgicels
Absorbable sponges
Povidone iodine
Nu Gauze sponges
Combine dressing
Dacron tube graft
Atraumatic partially occluding vascular clamps
Valved conduit (St. Jude Medical)
EVALUATING PROSTHETIC HEART VALVE IN SHEEP
New Methodologies
tus position, and prepped and draped in sterile fas-
hion. A left lateral thoracotomy was performed
through the 5th intercostal space. The descending
thoracic aorta was carefully exposed. The poste-
rior spinal arteries were isolated, so not to be in-
corporated later into the partially occluding aortic
clamp.
An intravenous bolus of unfractionated heparin
sodium (150 U/kg, Elkins-Sinn, Inc., Cherry Hill,
NJ) was given at this time. After 5 min, the descend-
ing aorta was partially cross-clamped distal to the
left subclavian artery using atraumatic, partially oc-
cluding vascular clamps. A valved conduit housing
a bilea et tilted disk (number 23 St. Jude Medical,
St. Jude Medical, Inc., St. Paul, MN) valve, with
an appropriately sized portion of woven, gelatin-
impregnated, tube graft (Gelweave, Sulzer Vascutek
USA, Inc., Austin, TX) sutured to the back end, was
anastomosed end-to-side to the isolated segment of
the proximal descending thoracic aorta (Figure 1).
The end-to-side anastomosis was performed at a 30
to 45 degree angle to minimize turbulence [17]. After
the anastomosis was completed using 4-0 mono la-
ment Prolene ( Johnson & Johnson Co., Somerville,
NJ) sutures, the partial aortic cross-clamp was reposi-
tioned to the supradiaphragmatic area for the distal
anastomosis, while the valved conduit was temporar-
ily clamped. Following completion of this anasto-
mosis, the aortic clamps were removed and ow was
established through the graft. This essentially cre-
ated a continuous tube graft in parallel with the de-
scending thoracic aorta with a centrally positioned
valve (Figure 2). Once adequate ow was con rmed
through the valved conduit, the native aorta was
Vaseline gauze Oxygen tank
Chest tube number 36 Fr Arterial line monitor
Endotracheal tubes Suction pump
Laryngoscope Ventilator
Sutures Capnometer
Heating pad Pulse oximeter
Bovie unit Pleurovac
Prep razors EKG leads/monitor
Gowns, caps, gloves Temperature probe
Umbilical tape Ultrasound machine
57
New Methodologies
FIGURE 1 Diagram of the customed tube graft with a cen-
trally located mechanical heart valve anastomosed to the de-
scending thoracic aorta with two partially occluding vascular
clamps in place.
ligated with surgical umbilical tape ( Johnson &
Johnson Co., Somerville, NJ). Absorbable oxidized
regenerated cellulose (Johnson & Johnson Medical
Inc., Arlington, TX) was applied to control bleeding
at the suture lines. After hemostasis was achieved,
a number 36 French chest tube (Argyle, Sherwood
Medical, St. Louise, MO) was placed through a sepa-
rate stab incision, connected to a pleurovac (Atrium
Medical Corp., Hudson, NH), and placed to low-
wall suction.
The thoracotomy incision was closed in a lay-
ered fashion. First, the ribs were reapproximated with
interrupted 0 Vicryl (Ethicon, Somerville, NJ) su-
ture. The muscle and fascia were closed in two layers
with running 2-0 Vicryl (Ethicon, Somerville, NJ)
suture. The skin was closed with interrupted 3-0 silk
58
(Ethicon, Somerville, NJ) suture. The animals was
then allowed to awake from anesthesia, and once
fully alert they were immediately extubated and the
arterial and venous lines were removed. The chest
tubes remained in place for a minimum of 2 h post-
operatively and were removed when the hourly chest
tube drain output had fallen to an acceptable level
and there was no air leak detected.
Postoperative Care
The animals continued their recovery in the vi-
varium sheep pens. For postoperative pain manage-
ment, meperidine (26 mg/kg, im, every 23 h;
Wyeth Laboratories, Inc., Philadelphia, PA) and
buprenorpherine (0.005 mg/kg, im, every 12 h;
Rickett &Coleman, Inc., Richmond, VA) were given
for 34 days. After this period, the narcotics were
withheld unless the sheep demonstrated signs of dis-
comfort or pain. The sheep received a 10-day course
of ampicillin sodium (7 mg/kg, sq) and gentamycin
(2 mg/kg, sq) that was administered twice a day.
There was no postoperative anticoagulation given.
Once the animals recovered completely from
surgery without ongoing complications and com-
pleted their postoperative antibiotic regimen, they
were placed in a transport cage and taken back to the
pasture farm.
Sacrice
The sheep remained on the farm until they re-
turned for sacri ce and explantation of the valves.
Sonographic evaluation of the valves and conduits
were performed prior to explantation. Following re-
moval of the valved conduits, the sheep were eutha-
nized with a lethal dose of Somelethal (J. A. Webster,
Inc., Sterling, MA).
RESULTS
The mean weight of the adult sheep was 33.8 kg
(range 24 to 43 kg). All of the sheep had a palpable
pulse in the conduit and an audible valvular click
prior to closure of the thoracotomy incision. It is
important to note that none of the sheep showed
M. M. PUC ET AL.

FIGURE 2 In situ photograph of the customed tube graft. The suture line indicates the position of the mechanical heart valve.
evidence of postoperative paralysis following a me-
dian partial aortic cross-clamp time of 39 min. In
addition, all the animals returned to the farm on
postoperative day 10 and were all noted to have an
audible valvular click.
Two sheep died shortly postoperatively (Table 3).
One death was attributed to intraoperative compli-
cations. In this case, the proximal anastomosis tore
posteriorly and had to be redone. However, the sheep
suffered a signi cant blood loss. Despite adequate
uid resuscitation, the sheep never fully recovered
from this insult and died 2 h postoperatively. The
second sheep died 4 h after extubation following an
TABLE 3 Postoperative deaths (n D 2)
Weight EBL Operative CC time
Animal (kg) (ml) time (min) Complications (min)
1 24 200 170 Arrhythmia
2 36 1000 165 Hypotension
Note. EBL, estimated blood loss; CC time, partial aortic cross-clamp
time.
EVALUATING PROSTHETIC HEART VALVE IN SHEEP
New Methodologies
uneventful perioperative course. It apparently died
from a cardiac arrhythmia. Prior to both of these
deaths, there were no air leaks appreciated from the
chest tubes in either sheep. Also, at necropsy, neither
animal had a signi cant hemothorax, and the valved
conduits were intact and patent with preserved valve
function.
The results of the four survivors are shown in
Table 4. These sheep had an uneventful operative
course and fully recovered postoperatively. They re-
turned to the pasture until the time of sacri ce. The
sheep returned for sacri ce and explantation of valves
at a median of 37 days (range 30 to 46 days). Sono-
graphic evaluation of the valves prior to sacri ce
showed no active movement of the lea ets in any of
the four sheep, but there was noted to be good blood
ow through the valved conduits. At explantation,
clot formation was found at the hinges of the lea ets
42
55 that had prevented active movement. The conduits
themselves were fully patent with intact proximal
and distal anastomotic sites.
59
New Methodologies
TABLE 4 Living sheep (n D 4 )
Animal Weight (kg) EBL (ml) OR time (min)
1. 43 150 270
2. 35 300 240
3. 31 200 170
4. 34 300 165
Note. EBL, estimated blood loss; OR time, total operative time; CT time, total time chest tube remained in place postoperatively; CC time, partial
aortic cross-clamp time; POD explant., postoperative day of explantation of valved conduit.
DISCUSSION
This study was initiated to establish a reliable in
vivo model for studying the thrombogenicity of
mechanical heart valves, since the currently avail-
able models did not meet our particular require-
ments [15, 16, 1821]. We had three main issues
that we wanted to adhere to: systemic circulation,
avoid cardiac bypass, and avoid complete aortic
occlusion.
In an attempt to minimize experimental variables,
we decided against placing the valve in the venous
circulation, as reported by Yang et al. in which a heart
valve was positioned within the inferior vena cava of
a sheep [21]. The different hemodynamic properties
that exist between the venous and arterial circula-
tions will affect the rate and pattern of thrombus for-
mation. For this reason, we wanted to place the valve
into the arterial circulation, to reproduce the true en-
vironment in which the valve will need to function.
Obviously, the ideal place is an intracardiac position.
However, we wanted to eliminate the need for car-
diac bypass and its associated risks of cerebrovascular
accidents, end-organ injury, and hemorrhagic com-
plications, to name a few. Chanda et al. has described
placing a bioprosthetic heart valve in the descending
thoracic aorta of juvenile sheep, which would satisfy
our rst two requirements but necessitates complete
aortic cross-clamping. Previous sheep studies in our
laboratory have shown that sheep have a poor toler-
ance for complete aortic occlusion. We experienced
sheep paralysis following total cross-clamp times of
as little as 16 min. Personal communications with
other investigators have witnessed sheep sustaining
paralysis following only 10 min of complete aortic
cross-clamp time. Our solution to this problem was
to only partially occlude the aorta with a side-biting
technique as illustrated in Figure 1. We were then
able to bypass the aorta with a tube graft that con-
60
CC time (h) Complications CC time (min) POD explant. (day)
18 Air leak 37 46
2 None 41 37
2 None 35 34
2 None 36 30
tained a mechanical heart valve without completely
occluding the aorta for any period of time. This tech-
nique allows adequate distal perfusion and does not
interrupt critical blood ow to the spinal cord. Our
median partial cross-clamp time of 39 min (range
35 to 55 min) produced no postoperative paralysis.
Even the sheep with a partial cross-clamp time of
55 min had no paraplegic sequela postoperatively
(Table 3).
The decision to use sheep was rather simple. Upon
review of the literature, sheep have a relatively large
aorta compared to the other animal models, such
as dogs, pigs, or calves. This makes sheep physi-
cally more appealing, along with their similar phys-
iologic parameters (e.g., heart rate and blood pres-
sure) as reported by Barnhart and associates [10].
More importantly, as suggested by Tillman and co-
workers, the coagulation pro le of sheep is similar to
that of humans [ 11]. This is critical when studying
the effects of thrombogenicity of mechanical heart
valves.
Overall, this study established a reliable and repro-
ducible animal model to enable continued research
in identifying a solution for eliminating the need
for lifelong anticoagulation with mechanical heart
valves.
CONCLUSION
This novel technique provides an acceptable alter-
native to the orthotopic placement of heart valves for
the study of innovative mechanical heart valve ma-
terials and designs. We developed an ef cient model
that enabled us to reach our endpoints. The mechani-
cal heart valve was placed within the systemic circula-
tion with avoidance of cardiac bypass and essentially
eliminated the risk of paraplegia. We strongly advo-
cate this model for studying the thrombogenicity of
mechanical heart valves.
M. M. PUC ET AL.

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61
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