Surface heparin treatment of the decellularized porcine heart valve:

Effect on tissue calcification

Min Yang,1 Yang-Hua Lin,1 Wei-Ping Shi,1 Hong-Can Shi,1 Y. John Gu,2 Yu-Sheng Shu1*
1

Department of Cardiothoracic Surgery, Northern Jiangsu Peoples Hospital Affiliated to Yangzhou University,
Yangzhou, Jiangsu Province, China
2
Department of Cardiothoracic Surgery, University Medical Center Groningen, Groningen, The Netherlands
Received 1 November 2014; revised 15 June 2015; accepted 2 July 2015
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33490
Abstract: Tissue calcification is a major cause of failure of
bioprosthetic heart valves. Aim of this study was to examine
whether surface heparin treatment of the decellularized porcine heart valve reduces tissue calcification. Fresh porcine
aortic heart valves were dissected as tissue discs and divided
into four groups. Group A: controls without treatment, Group
B: decellularization only, Group C: decellularization and glutaraldehyde cross-linking, Group D: decellularization and glutaraldehyde cross-linking followed by surface heparin
treatment. After implantation in New Zealand White rabbits
for 60 days, the explanted heart valve discs from the different
study groups underwent a series of histological examinations
as well as determination of calcium content by the methyl
thyme phenol blue colorimetric method. Results of the
explanted heart valve discs for the Von Kossa staining demonstrated that in Group A the heart valve tissue was the

most severely stained with black color, whereas in Group D

there was hardly any area that was stained black after
implantation indicating the least tissue calcification. Furthermore, the inflammatory cells identified by the Hematoxylineosin staining appeared to be the least in Group D. The average tissue calcium content was highest in Group A
(0.197 6 0.115 lmol mg21), modest in Group B (0.113 6 0.041
lmol mg21), and Group C (0.089 6 0.049 lmol mg21), and the
lowest in Group D (0.019 6 0.019 lmol mg21, p < 0.05). These
results suggest that surface heparin treatment tends to
reduce tissue calcification of the dellellularized porcine heart
C 2015
valve in a rabbit intramuscular implantation model. V
Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B:
000000, 2015.

Key Words: heparin, heart valve, calcification

How to cite this article: Yang M, Lin Y-H, Shi W-P, Shi H-C, Gu YJ, Shu Y-S. 2015. Surface heparin treatment of the
decellularized porcine heart valve: Effect on tissue calcification. J Biomed Mater Res Part B 2015:00B:000000.

INTRODUCTION

Despite recent advances of research and development in the

bioprosthetic heart valves, tissue calcication is still a major
problem in causing bioprosthetic valve failure in the
body.13 One of the reasons resulting in valve tissue calcication is believed to be the glutaraldehyde xation because
this cross linking process is known to promote the interaction between the free aldehyde groups, phospholipids, and
residual antigenicity of the bioprosthetic tissue.3,4 Although
several preventive methods have been developed to inhibit
the tissue calcication process,59 little has been seen as
successful application in combating the calcication problem
encountered after implantation.
Decellularization of the bioprosthetic heart valves is a
well-accepted method in manufacturing the tissueengineered heart valves.912 The advantage of decellularization is the complete removal of cells along with their antigenic cellular element.9 However, the drawback is that the
decellularization process per se removes all the endothelial
cells that are less likely to be reseeded by the adjacent tissue or circulating cells.13,14

Surface heparin treatment of the decellularized xenografts has been found to have an antithrombogenicity as
well as an antiproliferative property.15,16 Moreover, earlier
experiments have also indicated that surface heparin coating
may have an anticalcication effect on the glutaraldehydexed bioprosthetic heart valve.1719 Heparin is a glycosaminoglycan consisting of many sulfated repeating disaccharide
units. Because of its highly negative electric charge characteristics heparin can be covalently bound to the carboxy
side of matrix proteins such as collagen and elastin even
after decellularization.20,21 Thus, the present study was
undertaken to investigate whether surface heparin treatment has an inhibitory effect on tissue calcication of the
porcine heart valve that had undergone the process of both
glutaraldehyde xation and decellularization.
MATERIALS AND METHODS

Preparation of porcine heart valves

Sixty porcine aortic heart valves were obtained from adult
pigs in a local abattoir. They were then dissected into tissue
discs of 8 mm in diameter and rinsed in Hanks balanced

Correspondence to: Dr. Y.-S. Shu; e-mail: 18051062200@163.com

C 2015 WILEY PERIODICALS, INC.

V

salt solution (HBSS, Biochrom, Berlin, Germany) at 48C.

Warm ischemic time was <2 h. The heart valve discs were
then randomly divided into four groups. Group A: control
valve discs without treatment, Group B: the valve discs
were decellularizated by the detergent extraction and enzymatic method, Group C: the valve discs were rst decellularizated and then treated by glutaraldehyde cross-linking,
Group D: the valve discs were rst treated by decellularization and glutaraldehyde cross-linking and then treated by
surface heparin.
Decellularization
The heart valve discs in groups B, C, and D were placed,
respectively, in hypotonic and hypertonic Tris(hydroxymethyl) aminomethane Hydrochloride (Tris-HCL, Sigma, St.
Louis, MO) with 0.02% ethylenediamine tetraacetic acid
(EDTA, Gibco, Grand Island, NY) at 48C for 24 h. Afterward,
the heart valve discs were agitated in Tris-HCL with 1%
toctyl-phenoxypolyethanol (TritonX-100, Sigma) and 0.02%
EDTA at 48C for 24 h. These procedures were followed by
incubation with 20 mg mL21 ribonuclease A (RNase A,
Boehringer, Mannheim, Germany) and 0.2 mg mL21 deoxyribonuclease I (DNase I, Boehringer, Mannheim, Germany) at
378C for 2 h. The samples were subsequently placed in TrisHCL with 1% Triton X-100 at 48C for 24 h. All steps were
conducted in a 5% CO2/95% atmosphere under continuous
shaking. Finally, all these decellularized heart valve discs
were washed for ve times with PBS for 15 min each to
remove residual substances.
Glutaraldehyde xation
The decellularized heart valve discs in both groups C and D
were rinsed with 50 mL distilled water for three times and
then immersed into 0.625% glutaraldehyde (GA) at 328C for
72 h. Afterward, the heart valve discs were rinsed by with
50 mL distilled water for three times and immersed into
0.5% lysine solution for 42 h to keep the biocompatibility
during glutaraldehyde treatment.
Surface heparin treatment
The decellularized heart valve discs in group D were treated
by heparin immobilization by incubation in 1M hydroxylamine sulfate salt (Sigma) at room temperature on an orbital
shaker for 12 h. These heart valve discs were then rinsed in
distilled water and immersed into heparin and N-(3-dimethylaminoporpyl)-N0 -ethylcarbodiimide solution (heparin-EDC,
1.67-g EDC[Sigma]10.835-heparin sodium salt [Sigma]1
200-mL0.05-M HCL, PH1.5, prepared in our laboratory) on an
orbital shaker for 48 h.
Sterilization of the samples
After being packaged in double-foil, all of the heart valve
discs from the group A, B, C, D were irradiated with 60Co
25kGy (Shanghai Institute of Nuclear Research, Chinese
Academy of Sciences) for 15 min and they were stored in
HBSS at 48C.

YANG ET AL.

Animal experiments for valve implantation

Twelve male New Zealand White rabbits aged 68 months
were used in this study for valve implantation. The rabbits
were housed individually and received humane care in compliance with the Guide for the Care und Use of Laboratory
Animals published by the National Institutes of Health (NIH
publication No. 8523, revised 1985). Before the implantation experiments, each rabbits condition and health was
evaluated by a trained animal care technician. Each rabbit
was assigned a number, randomized by using Excel (Microsoft, Redmond, WA), and weighed. At implantation, and rabbits weighed 1.52.0 kg. All surgery was performed by
using aseptic techniques. Anesthesia was induced by peritoneal injection using ethyl urethane (750 mg kg21, Shengong,
Shanghai, China). For the convenience of operation, an incision was made on the right back of each rabbit through the
skin and into the latissimus dorsi muscle. Then, the four
heart valve discs respectively from the four groups were
inserted in the same muscle layer in a clockwise order without randomization. The incision was then sutured and bandaged. The signs of distress and pain, diet, wound healing
of the rabbit were observed every day after operation. After
60 days, the rabbits were euthanized by injecting 50 mL air
into their ear vein in accordance to the institutional protocol guidelines. The implanted heart valve discs were then
harvested (named group Ap, Bp, Cp, Dp) by an incision
made in the original skin area.
Histological examination
Heart valve tissue samples (n 5 5) from each of the group
A, B, C, D and the group Ap, Bp, Cp, Dp were cut into pieces
of 3 mm 3 3 mm and xed in 10% neutral buffered formalin for 24 h. Subsequently, these tissue blocks were embedded with parafn and cut into sections of 5 lm in thickness.
The hematoxylin-eosin staining was applied to observe the
cells within the heart valve tissues before and after implantation. The toluidine blue staining was used to conrm the
surface heparin coating. The implanted heart valve discs
harvested from the rabbits (group Ap, Bp, Cp, Dp) were
stained with Von Kossa silver staining and calcication was
assessed by calculating the number of calcied points
within 30 high magnication views. Then, an average number was presented as the mean 6 standard deviation.
Calcium determination
After explantation, the heart valve discs from groups Ap, Bp,
Cp, Dp (n 5 6) were put into a drying box for a constant
temperature of 1208C for 48 h. Then, the dry heart valve
discs were homogenized by adding 400 lL physiological
saline to each of the dry samples respectively. Afterwards,
the valve tissue homogenates were centrifuged for 10 min
(2500 RPM) to obtain the supernatants(25 ll). The calcium
content from the valve tissue was determined by the methyl
thyme phenol blue colorimetric method using an ultraviolet
spectrophotometer (Eppendorf.com, Germany) at a wavelength of 610 nm. Finally, the standard curve was drawn
and the calcium content was calculated.

SURFACE HEPARIN TREATMENT OF THE DECELLULARIZED PORCINE HEART VALVE

ORIGINAL RESEARCH REPORT

FIGURE 1. Hematoxylin-eosin staining of the porcine heart valve discs before and after implantation in rabbits for 60 days (magnification 3400).
Before implantation, there were only cells seen in the control heart valve tissue in (A), while there were hardly any cells seen in the heart valve
discs that had been decellularizated (B, C, and D). After implantation, the cells in the control heart valve discs were primarily accumulated on
the surface (Ap). In the decellularizated (Bp) and the decellularizated plus glutaraldehyde cross-linking heart valve discs (Cp), there were a large
number of newly migrating cells, likely cellular infiltrates, located both on the surface and inside the tissue. In the heart valve discs pretreated
with heparin (Dp), however, there were hardly any cells that could be found after implantation for 60 days. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]

Statistics
Data are reported as mean 6 standard deviation. Differences
among groups were analyzed by single-factor analysis of
variance using SPSS16.0. A p values <5% is considered
signicant.

ides [Figure 2(A)]. In group B and C, however, there were

little color visible and no mucopolysaccharides was available in the tissue layers [Figure 2(B,C)]. In group D, on the
contrary, the color of the toluidine blue staining was dark
and there were mucopolysaccharides distributed in all the
tissue layers [Figure 2(D)].

RESULTS

HematoxylinEosin staining
Before implantation, there were a great number of cells
remaining in the heart valve tissue and the bers of tissue
were wavelike and compact in group A [Figure 1(A)]. In
groups B, C, and D, on the contrary, there were no cells
remaining in the tissue and the bers of tissue were kept
wavelike and compact [Figure 1(BD)]. After implantation
for 60 days, there were cells distributed primarily on the
surface of the heart valve tissue in group Ap [Figure 1
(Ap)]. In group Bp, there were even more cells, likely the
cellular inltrates located both on the surface and inside the
tissue [Figure 1(Bp)]. In group Cp, however, there were only
a small number of cells (cellular inltrates) located both on
the surface and inside the tissue [Figure 1(Cp)]. In group
Dp, there were almost no cells that could be found both on
the surface and inside the heart valve tissue [Figure 1(Dp)].
Toluidine blue staining
The toluidine blue staining of the heart valve tissue showed
that in group A the color of the staining was dark and the
fresh tissue layers were fully lled with mucopolysacchar-

Von Kossa staining

The results of Von kossa silver staining showed that in the
Ap group the heart valve tissue was the most severely
stained to the black color, which indicated the severe tissue
calcication [Figure 3(Ap)]. In the Bp group, there were
many regions that were stained black [Figure 3(Bp)],
whereas in the Cp group, there were only some regions of
the tissue that were stained black [Figure 3(Cp)]; In the Dp
group, however, there was the least area that was stained
black, indicating that the tissue calcication is the most
mild [Figure 3(Dp)]. The average number of calcied points
found in the explanted heart valve tissue was 102.30 6
17.17, 68.20 6 7.05, 31.20 6 2.94, and 15.10 6 2.02, respectively in the Ap, Bp, Cp, and Dp groups (p < 0.05 between
Dp and any other groups).
Calcium contents
The average dry weight of the heart valve disc tissue was
1.0 6 0.4 mg, 1.7 6 1.2 mg, 1.6 6 1.2 mg, and 1.1 6 0.3 mg,
respectively in group Ap, Bp, Cp, and Dp. The concentration
of calcium content was calculated from the standard curve

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2015 VOL 00B, ISSUE 00

FIGURE 2. Toluidine Blue staining of the heart valve tissue before implantation (magnification 3400). Note that in group A the color of the staining is dark indicating that the fresh tissue layers were fully filled with mucopolysaccharides (A). In group B and C, there is little color visible as a
result of decellurarization (B and C). In group D, the toluidine blue staining reflects massive mucopolysaccharides distributed in all the tissue
layers (D) indicating effective heparin treatment. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

made according to the instruction of commercially available

calcium calorimetric assay kit. After correction by the
weight, the average tissue calcium content in group Ap, Bp,
Cp, and Dp was respectively 0.197 6 0.115 lmol mg21,
0.113 6 0.041 lmol mg21, 0.089 6 0.049 lmol mg21, and
0.019 6 0.019 lmol mg21 (P < 0.05, Figure 4).
DISCUSSIONS

Tissue calcication of the bioprosthetic heart valve is known

to be a major cause resulting in valve failure after implantation. We observed in this experimental study that after
implantation in rabbits for 60 days, porcine aortic valves
pretreated with surface heparin resulted in signicantly less
calcium accumulation in the valve tissue as demonstrated
by the least black color dye in the Von Kossa staining and
the lowest calcium contents by the methyl thyme phenol
blue colorimetric test. Moreover, there was a signicantly
less cell inltration in the porcine aortic valves pretreated
with heparin, suggesting that these results are in line with
our previous ndings of surface heparin treatment on the
decellularized xenograft tissues.15,16
There have been several techniques and methods developed to prevent the tissue calcication problem of the bioprosthetic heart valves.5,7,8 However, none of these methods
have been applied clinically as these used substances may
not be sufcient to block the whole calcication process.

Surface heparin treatment of the bioprosthetic heart valves

has been considered as a preventive method to inhibit tissue calcication.1719 These prior work involve both the
porcine pericardial bioprostheses and bovine pericardium.
Heparin is a glycosaminoglycan consisting of many sulfated
repeating disaccharide units. Because of its highly negative
electric charge characteristics heparin can be covalently
bound to the carboxy side of matrix proteins such as collagen and elastin even after decellularization.20,21 A potential
advantage of surface heparin treatment over the other anti
calcication methods is the possibility that the additional
heparin may compensate to the loss of glycosaminoglycan
during glutaraldehyde crosslinking.22 Previously, we have
reported that surface heparin treatment of the decellularized xenografts has an antithrombogenicity as well as an
antiproliferative property.15,16 In the current study, we
found that the porcine aortic valve tissue pretreated with
surface heparin contained signicantly less calcium than
those valve tissues without heparin pretreatment after 60
days implantation. Our results are in agreement with the
earlier experimental ndings on bovine pericardium pretreated with heparin reported by Lee et al.18 They
implanted both the heparin treated and glutaraldehyde
treated bovine pericardium in a rat subcutaneous model
and found that after 8 weeks there was signicantly less
calcium accumulation within the retrieved tissue in the

FIGURE 3. Von Kossa Staining of the porcine aortic heart valve discs following implantation in rabbits for 60 days (magnification 3400). In the
controls without pretreatment the heart valve tissue was the most severely stained with black color, indicating the severe tissue calcification
(Ap). In the decellularizated (Bp) and the decellularizated plus glutaraldehyde cross-linking heart valve discs (Cp), there were only some areas
that were stained black. In the heart valve tissues pretreated with heparin, however, there was least stained black, indicating the least tissue calcification (Dp). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

YANG ET AL.

SURFACE HEPARIN TREATMENT OF THE DECELLULARIZED PORCINE HEART VALVE

ORIGINAL RESEARCH REPORT

FIGURE 4. Calcium contents measured from different porcine aortic

heart valve discs after implantation in rabbits for 60 days. GA 5 glutaraldehyde, *p < 0.05 in comparison with other groups.

heparin treated group than that in the glutaraldehyde

treated group. This anticalcication effect of heparin is at
least obvious in the early stage of implantation, as observed
by the current study.
Glutaraldehyde xation is the most widely used crosslinking method for the manufacturing of the bioprosthetic
heart valves.23,24 The advantage of glutaraldehyde xation is
to improve the mechanical strength of the heart valve tissue
as well as to reduce immunogenicity. However, the drawback is that glutaraldehyde xation may potentially facilitate
tissue calcication. Under normal circumstances, the glycoproteins and other substances within the heart valve tissue
block the calcium phospholipid-binding sites. Glutaraldehyde xation results in the loss of these soluble proteins
and results in the formation of calcium phosphate precipitate that is believed to promote calcication. Furthermore,
the cross-linking reaction of glutaraldehyde with collagen
involves only the amino acid group rather than the hydroxyl
group thus leading to the exposure of the free hydroxyl
group of collagen to promote calcication. In the current
study, we covalently immobilized the heparin molecules
onto the glutaraldehyde-xed heart valve tissue before
implantation. Our in vivo observation clearly indicated that
the heart valve tissue that had undergone surface heparin
pretreatment had a signicantly lower calcium contents
than those glutaraldehyde-xed heart valve tissue without
surface heparin treatment. This study is the rst to report
the in vivo effect of surface heparin coating on
glutaraldehyde-xed bioprosthetic heart valve that has
undergone decellularization.
Although the mechanism with which heparin pretreatment reduces heart valve tissue calcication is not completely clear, it is likely that heparin molecules may
alternatively block the calcium phospholipid-binding sites
that have been inuenced by glutaraldehyde xation, a
mechanism previously proposed in the literature.1719 However, since heparin also has an antigrowth and antiproliferation effect,25 it may also inuence the growth pattern of
small muscle cells during the implantation period and indi-

rectly inhibit tissue calcication.18 Another issue concerning

the mechanism of surface heparin treatment on tissue calcication is the fact that during the heparin immobilization
process in vitro heparin had been xated with EDC, a carbodiimide that might have inuenced the effect of heparin on
tissue calcication in vivo. Nevertheless, it is necessary to
design further studies to pay special attention to the elucidation of the mechanisms of heparin pretreatment on tissue
calcication in bioprosthetic heart valves.
An ideal animal model in assessing the severity of in
vivo calcication of the bioprosthetic heart valve should
have a high sensitivity in detecting calcium production
within a relatively short period of time. For the current
study, we have chosen the New Zealand White rabbit intramuscular model as this model has been reported to be
superior to the commonly used Sprague-Dawley rat subcutaneous model for observing calcium accumulation in the
implanted bioprosthetic heart valve tissues.26 Compared
with the rat subcutaneous model, the rabbit intramuscular
model was associated with a shorter period of in vivo time
needed in detecting tissue calcication, less infection complications, larger area to implant bioprosthetic tissues
thereby reducing animal use numbers, and a more metabolically and mechanically dynamic environment than the rat
subcutaneous model.26 Another issue to optimize the sensitivity of calcium detection in vivo was the age of the animals. Younger rabbits demonstrate a relatively higher
calcium metabolism than their older counterparts as measured by their bone-building and hormonal regulation.27
Although the real effect of rabbit maturity on the calcium
metabolism is unknown, we did nd sufcient calcium content deposited in the control discs while the calcium deposition was signicantly reduced in the discs pretreated with
surface heparin. From the explanted heart valve discs a high
calcium content could be detected in the control discs
whereas only a very low calcium content was found in the
discs pretreated with surface heparin. These results conrm
the early ndings26 that the juvenile New Zealand White
rabbit is a sensitive experiment model in studying the in
vivo calcication problem in the bioprosthetic heart valves.
In conclusion, surface heparin treatment reduces the
in vivo calcication in the porcine heart valve that had
undergone decellularization and glutaraldehyde xation.
The rabbit intramuscular model is effective for the evaluation of tissue calcication of the implanted porcine heart
valves.
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SURFACE HEPARIN TREATMENT OF THE DECELLULARIZED PORCINE HEART VALVE