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Experimental studies on glycerol preserved vascular allografts
Fahner, Peter
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Fahner, P. J. (2014). Experimental studies on glycerol preserved vascular allografts
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Download date: 18 Jun 2017
4
Glycerol preserved arterial allografts
evaluated in the infrarenal rat aorta
Eur Surg Res 2009;42:78-86
P.J. Fahner1, M.M. Idu1, T.M. van Gulik1, B. van Wijk1,
A.C. van der Wal2, D.A. Legemate1
Department of Surgery and 2Department of Pathology
Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
1
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Abstract
Background. Vascular transplantation has become an alternative for prosthetic grafts. Suitable
storage methods for vascular allografts are therefore necessary. For small calibre arterial
allografts, cryopreservation and cold storage showed discouraging results. Since glycerol
preservation proved effective for the storage of skin allografts, this preservation method was
investigated for vascular allografts using a rat aortic transplantation model.
Methods. Glycerol preserved allografts (GA) were transplanted to the infrarenal aorta (n=18) in
Wistar rats. A control group (n=18) underwent immediate auto transplantation (AU) of an equal
length of aorta.
Results. Cumulative graft patency at 90 days follow-up was 93% for AU and 78% for GA (ns).
No aneurysm formation was detected in both groups. Intraluminal endothelial cell coverage,
integrity of the media and smooth muscle cell repopulation were comparable in both groups.
Intimal thickness was less in GA compared to AU and inflammatory reaction in the adventitia
was diminished in GA.
Conclusion. Glycerol preserved allografts were successfully grafted with an acceptable patency
rates if compared with autografts, while intima hyperplasia and adventitial inflammatory reaction
were less.
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Introduction
Transplantation of small diameter allografts is an alternative for patients who need an infrainguinal
arterial bypass graft, especially if autologous veins cannot be used. Vascular allografts require
proper storage techniques to ensure instantaneous availability. Owing to lower or absent
antigenic potential, preserved vascular allografts are by far superior to fresh allografts. Fresh
Brown Norway rat aorta segments induced a higher inflammatory reaction when subcutaneously
implanted in Lewis recipients compared to cryopreserved and glutaraldehyde preserved segments.
Cryopreserved aorta segments were stored for two weeks and glutaraldehyde segments for
three days before implantation (1). Different preservation methods such as cryopreservation,
glutaraldehyde tanning and cold storage have been the focus of extensive research for several
decades (2). Allograft function is hampered by antigenicity and graft rejection which can lead to
graft dilatation, intimal cell proliferation and graft rupture. Studies on cold-stored allografts have
4
been unsuccessful, due to disintegration and rapid rejection of the graft material. In the study
of van Reedt Dortland et al (3), cold-stored venous homografts, denatured for at least 6 weeks,
were used in femorodistal arterial reconstructions in patients when a suitable autologous vein was
not available. These grafts developed aneurysms in 58% after 5 years. Rebane et al performed
infrainguinal reconstructions for limb salvage in 107 patients. The venous allografts were cold
stored up to 10 days. Early thrombosis, indicating acute rejection, occurred in 16% and 5 year
graft patency rate was only 20% (3;4). Cryopreservation on the other hand has been extensively
used as a preservation method in the clinical setting, but mainly for large calibre arterial vessels
such as aortic allografts. Long-term patency of small calibre cryopreserved vascular grafts is poor
with reported patency rates of less than 50% at one year (5-7). Therefore, better preservation
techniques are necessary to improve the patency rate of small diameter vascular allografts.
Research on preservation techniques of skin allografts (8-10) showed that glycerol preservation
is superior to cryopreservation in regard to graft acceptance and immunogenicity. Glycerol skin
allografts, with a storage life of at least 2 years, were evaluated in 39 patients with extensive
third-degree burns. Cryopreserved allografts failed before epithelialisation was established
and glycerol allografts results in a complete graft take in 73% at one week postoperative. If
glycerol allografts were applied, wound epithelialisation by autologous epithelium was enhanced
compared to fresh-frozen allografts (11). Glycerol preservation of skin allografts attenuates
allograft reaction and has antiviral and antibacterial properties (12-17). The fact that glycerol
preservation results in a tissue matrix without living cells diminishes immunogenicity of the graft.
Previous experiments in our laboratory demonstrated that glycerol preservation of the rat aorta
preserves the biomechanical characteristics and architecture of the vessel wall (18).
The aim of this study was to evaluate in an aortic transplantation model in the rat i) long-term
patency of glycerol preserved arterial allografts in the infrarenal aortic position ii) aneurysmal
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degeneration of glycerol preserved arterial allografts, iii) histological features of the glycerol
preserved vessel wall after implantation, with emphasis on restoration of endothelial cell lining,
inflammatory response, degenerative changes of the vessel wall and intimal hyperplasia.
Materials and methods
Preparation of glycerol preserved allografts
The glycerolization protocol consists of three stages;
1. Incubation in a glycerol 50% solution for 4 hours at room temperature
2. Incubation in a glycerol 70% solution for 3 hours at 33°C
3. Incubation in a glycerol 85% solution for 3 hours at 33°C
After completion of the protocol, the grafts were stored in glycerol 85% for 12 hours at 4°C.
Previous in-vitro experiments in our laboratory showed that this protocol resulted in optimal
glycerol preservation of arterial allografts (18).
Experimental design
The experimental protocol was approved by the Animal Ethics Committee of the Academic
Medical Centre, University of Amsterdam, The Netherlands. Male outbred Wistar rats (Unilever
and Harlan), 300-370 g, were used as donors and recipients of aortic grafts (Charles Rivers,
Maastricht, The Netherlands). The animals were housed one week prior to the experiments,
had free access to water and chow and maintained on a 12-hour light-dark cycle. All operative
procedures were performed under clean but not sterile conditions. In the experimental (allograft)
group a 10 mm long segment of the infrarenal abdominal aorta was excised and a donor glycerol
preserved abdominal aortic allograft of similar length was implanted as an interposition graft. In
the control (autograft) group a segment of 10 mm of the infrarenal abdominal aorta was excised
and immediately reanastomosed to restore aortic continuity. Before implantation the glycerol
preserved aortic allograft was immerged in saline to diminish the tissue content of glycerol in the
graft at 33°C for at least 20 minutes. Both control and experimental groups consisted of eighteen
rats. Scheduled sacrifice was on post-operative day 1, 3, 7, 14, 30 and 90 respectively. On these
days three animals were sacrificed in both experimental and control group.
Aortic allograft implantation and harvesting
All animals were anesthetized by inhalation of a mixture of O2/N2O 1:1 and isoflurane 0.8 - 2.0%.
After endotracheal intubation the rats were ventilated and anaesthesia was maintained with
the same mixture. The rats were placed on a heating pad and positioned under a heating lamp
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to maintain a rectally measured core temperature between 36°C and 37°C. After a midline
laparotomy, the infrarenal abdominal aorta was exposed, clamped and a 10 mm segment was
excised using an operation microscope (Zeiss™, Germany). The grafts and the aortic stumps were
flushed with saline. No heparin or anticoagulant medication was used in the study. The endto-end anastomoses were performed with interrupted 9.0 sutures (Ethylon™). After restoring
the blood flow intra-operative patency was determined by visualization of a pulse distally to
the graft. Operation time and aortic clamping time averaged 90 and 45 minutes, respectively.
After sacrifice of the rat the graft was harvested en-bloc, flushed with saline and fixed in 10%
formaldehyde.
Duplex scanning and angiography
To assess graft patency and aneurysm formation, defined as a consistent 50% increase in graft
diameter, colour duplex scanning (19) and angiography were performed. Duplex scanning
4
(Hewlett Packard Sonos 5000) was performed under general anaesthesia using a miniature
15 MHz probe at post-operative day 1, 3, 7, 14, 30 and 90 respectively. The abdominal aorta was
examined from the renal arteries to the aortic bifurcation. Diameters of the aorta were measured
in B-mode on the following five locations; proximal native vessel, proximal anastomosis, midgraft, distal anastomosis and distal native vessel. To investigate the presence of flow-limiting
lesions peak systolic velocity (PSV) was measured. The PSV-max was defined as the maximum
peak systolic velocity in a stenosis, and the PSV-ratio as the PSV in the stenosis divided by the PSV
in the pre- or poststenotic region. A significant stenosis was defined as a PSV-ratio of ≥ 2.5 or a
PSV-max of ≥ 250 cm/sec. Before excision of the aortic allograft a digital subtraction angiography
(DSA) was performed (Philips™) by injection of three ml radio contrast solution (Visipaque™
320 mg I/ml, Nycomed, The Netherlands) through a catheter introduced into the carotid artery.
Preparation and histological staining of specimens
All explanted graft segments were flushed with 10% buffered formaldehyde, dehydrated and
embedded in paraffin for light microscopic examination. Grafts were divided into three equal
segments of 3-4 mm length: one proximal segment including the proximal anastomosis, one
midgraft segment and one distal segment including the distal anastomosis. Of each segment,
5 µm sagittal sections were cut and stained with haematoxylin-eosin (H&E), picosirius red (PSR)
and Elastica van Gieson (EvG) respectively. PSR stains all types of collagen bright red and EvG
stains elastic fibres deep black. Two additional sections were mounted for immunohistochemistry.
We used Anti-vWF (von Willebrand factor) antibody (DAKO, dilation 1: 100, visualizes all
endothelial cells) and anti-alpha-actin-antibody (DAKO, dilution 1: 400, visualizes vascular smooth
muscle cells at all stages of maturation) as primary antibodies. For detection of immunoreactivity
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we applied a streptavidin biotin complex method with DAB as substrate. Fresh full thickness
arterial wall was used as positive control tissue.
Histopathologic evaluation of grafts
Endothelial cell lining, integrity of elastin meshwork of the media, presence of medial smooth
muscle cells and adventitial inflammation were assessed on a semi quantitative basis. Two
observers (PJF and BvW) independently scored all sections which were blinded for study group
and follow-up period.
Morphometry
For morphometric analyses we used Image Pro-4 software (Image Pro-plus 4.5) (20). The
specimens were evaluated with a 10 times magnification objective of the light microscope
(Olympus, BX60). Intima thickness (distance between inner surface and internal elastica lamina)
and media thickness (distance between inner and outer elastica lamina) were automatically
measured at 6 places in each specimen and mean values were calculated.
Collagen content of the media and adventitia were quantified planimetrically on Picosirius
red stains for which we used grey scale detection with fixed threshold.
Statistical analysis
All statistical analyses were performed with GraphPad Prism 4.00 programme (GraphPad
Software, San Diego, USA). For comparison of cumulative graft patency data the log rank test
was used. Differences between groups in the morphological and morphometric analyses were
tested with Student-t test for continuous data and with the Mann-Whitney U test for comparison
of the semi-quantitative scores. The Kruskal-Wallis test was used to compare more than two
unpaired groups. A p-value of less than 0.05 was considered statistical significant.
RESULTS
Animal survival and graft patency
All animals survived the postoperative period until scheduled sacrifice, except one in the glycerol
group which was sacrificed at day 6 postoperatively because of severe weight loss. Macroscopic
examination of the harvested aorta revealed occlusion of the allograft. Of all other animals,
4 grafts occluded, one in the control group and three in the glycerol group. All occlusions
occurred within the first 3 postoperative days as was determined by duplex scanning. Cumulative
graft patency at 3 months follow-up for the autografts and the glycerol preserved allografts was
93% and 78% respectively (p = 0.14).
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mean PSV at day 7(cm/sec)
175
150
125
100
75
50
25
0
PNal
PNau
PAal
PAau
MGal MGau
DAal
DAau
DNal
DNau
localization of measurement in native vessel and graft
Figure 1. Mean peak systolic velocities (cm/sec) of glycerol allografts and autografts after 7 days follow-up.
Error bars depict SEM. PN = proximal native vessel, PA = proximal anastomosis, MG = midgraft, DA = distal
anastomosis, DN = distal native vessel, al = glycerol allograft, au = autograft. (MGal and MGau; p< 0.01).
mean PSV at day 90 (cm/sec)
= autograft = glycerol allograft
4
130
120
110
100
90
80
70
60
50
40
30
20
10
0
PNal
PNau
PAal
PAau
MGal
MGau
DAal
DAau
DNal
DNau
localization of measurement in native vessel and graft
Figure 2. Mean peak systolic velocities (cm/sec) of glycerol allografts and autografts after 90 days followup. Error bars depict SEM. PN = proximal native vessel, PA = proximal anastomosis, MG = midgraft, DA =
distal anastomosis, DN = distal native vessel, al = glycerol allograft, au = autograft. (PNal and PNau; p< 0.01).
= autograft = glycerol allograft
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Duplex sonography and angiographic parameters
The mean PSV at the different sites of the graft in both groups are represented in figure 1 and
2. Two autografts and one allograft showed a significant stenosis. In the midgraft part of the
transplanted allografts the mean PSV after 7 days follow-up was significantly lower compared
to the autografts (p< 0.01). No significant difference was detected after one months and after
3 months follow-up the mean PSV was consistently lower in the allografts at all locations although
only significantly different in the proximal native vessel in the transplanted allografts (p< 0.01).
No aneurysm formation or graft-disintegration was found during follow-up. After 7 and
90 days follow-up no significant difference in graft diameter was measured between both
graft types. After one month the mean diameter of the allografts was significantly higher at
the proximal anastomosis (p< 0.01) and midgraft (p= 0.02) as shown in figure 3. Percentage
graft stenosis on angiograms was not encountered in the midgraft segments, neither in the
allografts or autografts. In 4 autografts a stenosis was measured at the proximal anastomosis
and in 6 autografts at the distal anastomosis. Three glycerol allografts developed a stenosis at
the proximal anastomosis and one at the distal anastomosis. No significant difference existed
between autografts and allografts (p= 0.17).
Morphological and morphometric results
Intima
Endothelial cell (EC) coverage was assessed with anti-Vwf immunostaining. When pooling
the scores of the endothelial cell coverage at all time points for both graft types, intraluminal
endothelial cell coverage was more abundant in the proximal and distal segments compared to
the midgraft segments (p= 0.26). Endothelial cell coverage is presented in figure 4 in relation
with implantation time. There are no consistent differences in endothelial cell coverage between
autografts and glycerol grafts during the implantation period.
Intimal thickness was evaluated in the same segments using H&E stained sections. Increase in
intimal thickness, interpreted as intimal hyperplasia, was first observed in the glycerol allografts
at day 3 and in the autografts at day 14. When pooling the neo-intimal thickness of all graft
segments, intimal thickness was significantly greater in the autografts compared to the glycerol
preserved allografts after 1 and 3 months follow- up (figure 5, p< 0.01).
Media
The integrity of the elastic meshwork of the media, evaluated in Elastica van Gieson stained
sections, did not differ significantly between both types of grafts (p = 0.64). The length of
implantation period did not affect elastin network integrity which was comparable in all graft
segments.
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graft diameter at day 30 (mm)
3
2
1
0
PAal
PAau
MGal
MGau
DAal
DAau
4
localization in graft
Figure 3. Mean graft diameter measurements (mm) of allografts and autografts after 30 days follow-up.
Error bars depict SEM. PA = proximal anastomosis, MG = midgraft, DA = distal anastomosis, al = glycerol
allograft, au = autograft. (PAal and PAau; p> 0.01, MGal and MGau; p= 0.02).
= autograft = glycerol allograft
mean total score
4
3
2
1
0
gl1
a1
gl3
a3
gl7
a7
gl14
a14
gl30
a30
gl90
a90
follow up (days)
Figure 4. Mean score of intraluminal endothelial cells (Factor VIII staining) after pooling of results for
proximal, midgraft and distal segments. Error bars depict SEM. Score 0 = complete coverage, 1 = some
endothelial lining, 2 = no endothelial cells. gl = glycerol allograft, a = autograft.
= autograft = glycerol allograft
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60
intimal thickness (μm)
50
40
30
20
10
0
1
3
7
14
30
90
follow up (days)
Figure 5. Pooled results of proximal, midgraft and distal mean intimal thickness of allografts and autografts.
Error bars depicts SEM.
= autograft = glycerol allograft
Medial width in proximal, mid and distal graft segments was significantly less in the glycerol
allografts compared to the autografts (p<0.01). Figure 6a shows a mid segment of a glycerol
allograft and figure 6b shows a mid segment of an autograft. Differences were most prominent
after day-1 and day-3 postoperatively and diminished during further follow-up. The pooled mean
thickness (SD) was 52.2 (12.9) μm for glycerol allografts and 71.1 (27.3) μm for autografts.
In all segments, apart from scant mononuclear infiltration around the sutures, no inflammatory
reaction was observed in both graft types.
The pooled mean collagen content of the media was lower in autografts compared to
allografts at all time points. In the autografts a decrease of the mean collagen content was
measured in the first three days postoperatively which however recovered, and after 14 days
exceeded the amount at day-1. For the glycerol allografts collagen content remained almost
similar at all time points (figure 7).
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Figure 6A. Haematoxylin-eosin staining of a
glycerol allograft midgraft segment (10x).
Figure 6B. Haematoxylin-eosin staining of an
autograft midgraft segment (10x). Arrows point
to the media in both figures.
4
percentage collagen in media (%)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1
3
7
14
30
90
follow-up (days)
Figure 7. Results of pooled data of percentage collagen in media of glycerol allografts and autografts,
referring to proximal, midgraft and distal graft segments.
= autograft
= glycerol allograft
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7
*
mean total score
6
5
4
3
2
1
0
gl1
a1
gl3
a3
gl7
a7
gl14
a14
gl30
a30
gl90
a90
follow up (days)
Figure 8. Mean score of α-smooth muscle cell (smc) staining of medial cells after pooling of results for
proximal, midgraft and distal segments. Scores of all three segments were added and the mean calculated
(score for single segment; 0 = > 50% smc ingrowth, 1 = < 50% smc ingrowth, 2 = some smc, n=3 for each
follow-up). Mean maximal score is 3x2, mean minimal score is 3x0. * p= 0.01. Error bars depict SEM.
= autograft = glycerol allograft
Presence of smooth muscle cells in the extra cellular tissue matrix was evaluated with anti
α- smooth muscle actin immunostaining. Both graft types showed slightly lower numbers of
smooth muscle cells in the distal segments compared to the proximal and medial segments. As
expected, the highest amount of positively stained cells was found in the autograft segments
at day-1 postoperatively. In the glycerol allografts this amount diminished in the first week
postoperatively and recovered to almost the level of day three after three months follow-up
(figure 8). No consistent differences in smooth muscle cell repopulation between autografts and
glycerol allografts exist during follow-up.
Adventitial parameters
Quantification of collagen in the adventitia of both graft types and in the different graft segments
showed equal results.
Both graft types showed a higher score of inflammatory reaction at the sites of anastomosis.
The pooled score at the proximal and distal segments was lower for the glycerol allografts
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14
13
total score for inflammation
12
11
10
9
8
7
6
5
4
3
2
4
1
0
0
10
20
30
40
50
60
70
80
90
100
follow up (days)
Figure 9. Results of adventitial inflammation scoring in haematoxylin-eosin stained sections. Data shown
are the result of pooling of the proximal, medial and distal segments (
= allografts, ----- = autografts).
Score 0 = some lymphocytes, 1 = clusters of lymphocytes, 2 = diffuse infiltration.
compared to the autografts. This indicates a less extensive inflammatory reaction in the
glycerol allografts after implantation. The pooled score of the midgraft segments was slightly
lower in the allografts. The calculated total inflammatory score for proximal, medial and distal
segments reached its maximum earlier in glycerol grafts compared to autografts (day 14 and day
30 respectively). During follow-up a decrease in inflammation was measured in both groups,
inflammation being less in the allografts (figure 9).
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DISCUSSION
The infrarenal rat aortic replacement model is well established in the investigation of alternatives
for the venous or arterial autograft (21-24). It is clear that for the reconstruction of large arteries
synthetic vascular grafts (e.g. ePTFE or Dacron) are the conduits of choice. However, for small
diameter reconstructions such prosthetic grafts have low cumulative patencies, ranging from
24% to 58% after three years (25;26). These results have stimulated research in alternatives to
prosthetic grafts such as preserved vascular allografts and xenografts, and the use of biomaterials
to serve as a vascular tissue matrix (27;28). Extra cellular matrix scaffolds can serve as templates
for cellular attachment and ingrowth of recipient cells (29;30). Main efforts in this field have been
directed to minimizing immunogenicity and inflammation of the graft, while preserving extra
cellular matrix integrity and mechanical properties (31).
More details of the glycerol preservation process in skin allografts were clarified by Huang
et al and Ross et al. In the permeation of glycerol in skin a process of diffusion and binding is
involved. Both histological and ultra structural analysis showed that the integrity of skin structure
was maintained and degradation of the skin avoided due to effective sequestration of water
(32;33). The preservation of connective tissue after glycerolization was confirmed by glycerol
preservation of ovine cardiac valve allografts and mechanical properties maintained for 6 months
after implantation in the ovine descending aorta (34;35).
In the present study, glycerol preserved aortic allografts were examined as an alternative
biomaterial for arterial grafting. Patency rate after 3 months was 78% for glycerol preserved
allografts. These results are in accordance with the high patency rates after 100 days reported
by Wolff et al who transplanted rat aortic allografts and femoral veins preserved in 98% glycerol
(36). In the study of Wolff, however, no angiography or sonography was performed rendering
reliability of patency detection uncertain. Also possible aneurysm formation could have easily
been missed in this report. In our study aneurysm formation could be ruled out and owing to
the histological examinations new insight was provided into the performance of the glycerol
preserved allografts after implantation. The results of the present study and that of the study of
Wolff are different from the total graft occlusions after two weeks reported by Bishop et al (37).
In this study glycerol preserved DA strain rat femoral vein was transplanted into the common iliac
artery of Lewis rats. In the same study hind leg and foreleg veins were preserved in glycerol 98%
and transplanted to the common carotid artery. After six month of follow up a patency rate of
64% was reached. They reported a very strong allograft reaction as a result of the two rat strains
which obviously influenced patency rates.
Two pathways are involved in the immune response against allografts in transplant
vasculopathy. At first, donor endothelial cells and antigen presenting cells (APC) of the allograft
induces proliferation of recipient T- cells. Donor MHC I molecules on the surface of transplanted
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cells induces T- cell activation (38). Secondly, recipient APC’s present donor MHC and minor
antigens from the allograft to recipient T- cells. This requires recognition of the allogens as
peptides bound to recipient MHC II molecules (39;40).
Direct sensitization of the recipient by Langerhans cells, which have a strong expression
of MHC II molecules and are the APC’s of the skin, will be blocked in glycerol preserved skin
allografts since active migration of Langerhans cells from the allograft is no longer possible due
to cell death after glycerolization.
In the current experiments we used outbred Wistar rats. It could be debated if intimal
hyperplasia would have been more distinct when immunologically more different animal strains
had been used for our aortic transplantation experiments. Osako et al (41) demonstrated more
intimal hyperplasia in fresh and cryopreserved allografts compared to fresh and cryopreserved
isografts in their Lewis rat aortic transplantation model. However, Takeishi et al (42), who
performed cryopreserved femoral artery transplantation in Lewis and Brown Norway rats, found
4
quite similar patency rates for isografts (100% and 87%) and allografts (100% and 78%) after 1
and 3 months follow up.
Although it is difficult to assess the effect of genetically incompatibility between different
rat strains in relation to patency rates, one should be aware of the genetically relation when
comparing results of vascular implantation studies in which different rat strains were used. The
difference in above mentioned patencies is in agreement with the lower genetically uniformity
between DA and Lewis rats compared to BN and Lewis rat strains. This is confirmed by the higher
amount of cellular damage in DA donor livers compared to BN donor livers in an orthotopic rat
liver transplantation model (43).
The role of intraluminal endothelial cells is crucial in vascular graft patency. Several studies
have been performed to examine preservation methods for maintaining endothelial cell viability.
Preservation of luminal endothelial cell lining in University of Wisconsin (UW) solution appears
more successful compared to phosphate buffered saline (PBS) for up to seven days of cold storage
of rat carotid arteries (44). Longer periods of cold storage however led to endothelial cell death
and incomplete luminal coverage, which resulted in intimal hyperplasia and graft occlusion in
transplanted rabbit femoral arteries (45). Although on the one hand, the best thromboresistant
graft surface is luminal lining with host endothelial cells, transplanted donor endothelial cells on
the other hand, evoke an immunogenic response that will induce pathologic changes in graft
wall architecture (46;47). Furthermore, initial absence of allograft endothelium did not seem to
influence graft patency in the experiments of Komorowska et al (48) in which cryopreserved
femoral arteries were transplanted in a rat model. The absence of endothelium is probably
an advantage of glycerol preservation since no viable endothelial cells are transplanted while
the extracellular tissue matrix is used as template for recipient cell attachment and ingrowth
(49;50). Our study reveals endothelial cells in glycerol preserved allografts after transplantation
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which obviously originate from repopulating cells of the recipient. Theoretically this has the
advantage of the development of an antithrombotic luminal surface without the disadvantage
of immunologic reactions after transplantation of viable donor cells (51). Another important
finding is the lesser intimal hyperplasia as measured after one and three months and the lower
inflammatory response after one month follow-up in glycerol preserved allografts. Since intimal
hyperplasia is the common healing response to arterial wall injury and occurs subsequent to
immunoinflammatory endothelial injury, glycerol preservation has potential advantages (52).
The absence of living endothelial cells in the glycerol allografts unable those to induce the
cascade of cellular damage after transplantation which results in proliferation of smooth muscle
cells and fibroblasts involved in intimal hyperplasia. This will be valuable in the application of
glycerol allografts in humans since endothelialization is prolonged in humans (53;54). Although
the endothelialization of prosthetic grafts is at least 7.5 times more pronounced in any animal
model compared to human (including rat, dog and baboon models), interpretation of graft
endothelialization in rat models to humans is complicated by graft dimension difference (55).
In conclusion, in our aortic transplantation model in the rat, glycerol preserved allografts
were successfully grafted and had acceptable graft patencies if compared with autografts in
conjunction with diminished intimal hyperplasia and in the absence of aneurysmatic matrix
degeneration. These results warrant further investigation of this preservation method in a
clinically relevant, large animal model.
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Reference List
(1) Moriyama S, Utoh J, Sun LB, Tagami H, Okamoto K, Kunitomo R, et al. Antigenicity of
cryopreserved arterial allografts: comparison with fresh and glutaraldehyde treated grafts. ASAIO J
2001 May;47(3):202-5.
(2) Dardik H, Wengerter K, Qin F, Pangilinan A, Silvestri F, Wolodiger F, et al. Comparative decades
of experience with glutaraldehyde-tanned human umbilical cord vein graft for lower limb
revascularization: an analysis of 1275 cases. J Vasc Surg 2002 Jan;35(1):64-71.
(3) van Reedt Dortland RW, van Leeuwen MS, Steijling JJ, Theodorides T, van Vroonhoven TJ. Longterm results with vein homograft in femoro-distal arterial reconstructions. Eur J Vasc Surg
1991 Oct;5(5):557-64.
(4) Rebane E, Tikko H, Tunder E, Lepner U, Helberg A, Pulges A, et al. Venous allografts for infrainguinal
vascular bypass. Cardiovasc Surg 1997 Feb;5(1):21-5.
(5) Albertini JN, Barral X, Branchereau A, Favre JP, Guidicelli H, Magne JL, et al. Long-term results of
arterial allograft below-knee bypass grafts for limb salvage: a retrospective multicenter study. J Vasc
Surg 2000 Mar;31(3):426-35.
(6) Farber A, Major K, Wagner WH, Cohen JL, Cossman DV, Lauterbach SR, et al. Cryopreserved saphenous
vein allografts in infrainguinal revascularization: analysis of 240 grafts. J Vasc Surg 2003 Jul;38(1):1521.
(7) Martin RS, III, Edwards WH, Mulherin JL, Jr., Edwards WH, Jr., Jenkins JM, Hoff SJ. Cryopreserved
saphenous vein allografts for below-knee lower extremity revascularization. Ann Surg
1994 Jun;219(6):664-70.
(8) Brown JB, Fryer MP, Randall P, Lu M. Postmortem homografts as biological dressings for extensive
burns and denuded areas; immediate and preserved homografts as life-saving procedures. Ann Surg
1953 Oct;138(4):618-30.
(9) Robson MC, Krizek TJ. Predicting skin graft survival. J Trauma 1973 Mar;13(3):213-7.
4
(10) Schechter I, Belldegrin A, Ben Basat M, Kaplan I. Prolonged retention of glutaraldehyde-treated skin
homografts in humans. Br J Plast Surg 1975 Jul;28(3):198-202.
(11) Kreis RW, Vloemans AF, Hoekstra MJ, Mackie DP, Hermans RP. The use of non-viable glycerolpreserved cadaver skin combined with widely expanded autografts in the treatment of extensive
third-degree burns. J Trauma 1989 Jan;29(1):51-4.
(12) Basil A. A comparative study of glycerinized and lyophilized porcine skin in dressings for third-degree
burns. Plast Reconstr Surg 1982;69:969-74.
(13) de Backere AC. Euro Skin Bank: large scale skin-banking in Europe based on glycerol-preservation of
donor skin. Burns 1994;20 Suppl 1:S4-S9.
(14) Richters CD, Hoekstra MJ, van Baare J, du Pont JS, Kamperdijk EW. Morphology of glycerol-preserved
human cadaver skin. Burns 1996 Mar;22(2):113-6.
(15) van Baare J, Buitenwerf J, Hoekstra MJ, du Pont JS. Virucidal effect of glycerol as used in donor skin
preservation. Burns 1994;20 Suppl 1:S77-S80.
(16) van Baare J, Ligtvoet EE, Middelkoop E. Microbiological evaluation of glycerolized cadaveric donor
skin. Transplantation 1998 Apr 15;65(7):966-70.
(17) van Baare J, Cameron PU, Vardaxis N, Pagnon J, Reece J, Middelkoop E, et al. The 1998 Lindberg
Award. Comparison of glycerol preservation with cryopreservation methods on HIV-1 inactivation.
J Burn Care Rehabil 1998 Nov;19(6):494-500.
(18) Fahner PJ, Idu MM, Legemate DA, Vanbavel E, Borstlap J, Pfaffendorf M, et al. Morphological and
functional alterations in glycerol preserved rat aortic allografts. Int J Artif Organs 2004 Nov;27(11):97989.
Glycerol preserved arterial allografts evaluated in the infrarenal rat aorta | 73
Fahner.indd 73
5-5-2014 14:35:12
(19) Wessely R, Paschalidis M, Wagenpfeil S, Wegener F, Neumann FJ, Theiss W. A comprehensive
approach to visual and functional assessment of experimental vascular lesions in vivo. Am J Physiol
Heart Circ Physiol 2004 Jun;286(6):H2461-H2467.
(20) Blatt RJ, Clark AN, Courtney J, Tully C, Tucker AL. Automated quantitative analysis of angiogenesis
in the rat aorta model using Image-Pro Plus 4.1. Comput Methods Programs Biomed 2004 Jul;75(1):
75-9.
(21) Qin F, Dardik H, Pangilinan A, Robinson J, Chuy J, Wengerter K. Remodeling and suppression of
intimal hyperplasia of vascular grafts with a distal arteriovenous fistula in a rat model. J Vasc Surg
2001 Oct;34(4):701-6.
(22) Ribbe EB, Holmin T, Lowenhielm PC. Microvascular polytetrafluoroethylene (Gore-Tex) grafts in the
infrarenal rat aorta. Microsurgery 1987;8(2):48-53.
(23) Tizian C, Glass KD, Demuth R. Using expanded polytetrafluoroethylene for bridging arterial
microvascular defects: an experimental study with evaluation in the scanning electron microscopy.
Thorac Cardiovasc Surg 1980 Aug;28(4):273-6.
(24) Zelder O, Werner HH, Jerusalem CR. The use of microvascular graft as an arterial substitute in the
abdominal aorta of the rat. Microsurgery 1983;4(3):157-63.
(25) Eickhoff JH, Broome A, Ericsson BF, Buchardt Hansen HJ, Kordt KF, Mouritzen C, et al. Four years’
results of a prospective, randomized clinical trial comparing polytetrafluoroethylene and modified
human umbilical vein for below-knee femoropopliteal bypass. J Vasc Surg 1987 Nov;6(5):506-11.
(26) Johnson WC, Lee KK. A comparative evaluation of polytetrafluoroethylene, umbilical vein, and
saphenous vein bypass grafts for femoral-popliteal above-knee revascularization: a prospective
randomized Department of Veterans Affairs cooperative study. J Vasc Surg 2000 Aug;32(2):268-77.
(27) Hilbert SL, Ferrans VJ, Jones M. Tissue-derived biomaterials and their use in cardiovascular prosthetic
devices. Med Prog Technol 1988;14(3-4):115-63.
(28) Zdrahala RJ. Small caliber vascular grafts. Part I: state of the art. J Biomater Appl 1996 Apr;10(4):30929.
(29) Stansby G, Berwanger C, Shukla N, Hamilton G. Endothelial cell seeding of vascular grafts: status and
prospects. Cardiovasc Surg 1994 Oct;2(5):543-8.
(30) Williams SK, Schneider T, Kapelan B, Jarrell BE. Formation of a functional endothelium on vascular
grafts. J Electron Microsc Tech 1991 Dec;19(4):439-51.
(31) Schmidt CE, Baier JM. Acellular vascular tissues: natural biomaterials for tissue repair and tissue
engineering. Biomaterials 2000 Nov;21(22):2215-31.
(32) Huang Q, Pegg DE, Kearney JN. Banking of non-viable skin allografts using high concentrations of
glycerol or propylene glycol. Cell Tissue Bank 2004;5(1):3-21.
(33) Ross A, Kearney JN. The measurement of water activity in allogeneic skin grafts preserved using high
concentration glycerol or propylene glycol. Cell Tissue Bank 2004;5(1):37-44.
(34) Aidulis D, Pegg DE, Hunt CJ, Goffin YA, Vanderkelen A, Van HB, et al. Processing of ovine cardiac
valve allografts. Effects of preservation method on structure and mechanical properties. Cell Tissue
Bank 2002;3(2):79-89.
(35) Neves J, Abecassis M, Santiago T, Ramos T, Melo J, Gruys E, et al. Processing of ovine cardiac
valve allografts. Implantation following antimicrobial treatment and preservation. Cell Tissue Bank
2002;3(2):105-19.
(36) Wolff KD, Dienemann D. Vessel preservation with glycerol: an experimental study in rats. J Oral
Maxillofac Surg 1990 Sep;48(9):914-8.
(37) Bishop AJ, Glasby MA, Houlton JE. A morphological assessment of vein allografts preserved in
glycerol and used for arterial replacement. J Cardiovasc Surg (Torino) 1987 Sep;28(5):491-7.
74 | Chapter 4
Fahner.indd 74
5-5-2014 14:35:12
(38) Denton MD, Davis SF, Baum MA, Melter M, Reinders ME, Exeni A, et al. The role of the graft
endothelium in transplant rejection: evidence that endothelial activation may serve as a clinical
marker for the development of chronic rejection. Pediatr Transplant 2000 Nov;4(4):252-60.
(39) Watschinger B, Gallon L, Carpenter CB, Sayegh MH. Mechanisms of allo-recognition. Recognition
by in vivo-primed T cells of specific major histocompatibility complex polymorphisms presented as
peptides by responder antigen-presenting cells. Transplantation 1994 Feb 27;57(4):572-6.
(40) Zdoroveac A, Doebis C, Laube H, Brosel S, Schmitt-Knosalla I, Volk HD, et al. Modulation of Graft
Arteriosclerosis in a Rat Carotid Transplantation Model. J Surg Res 2007 Sep 29.
(41) Osako M, Otani H, Yamamura T, Nakao Y, Hattori R, Omiya H, et al. Alloimmune response
may be involved in neointimal hyperplasia in cryopreserved aortic allografts. Transplant Proc
2001 Jun;33(4):2566-70.
(42) Takeishi M, Hirase Y, Kojima T. Experimental study of cryopreserved allogeneic transfer of vessel:
preliminary report 4. Microsurgery 1994;15(1):55-62.
(43) Mollevi DG, Ginesta MM, Domenech P, Serrano T, Figueras J, Jaurrieta E. Hemostatic status in longterm surviving xenografts. Surgery 2006 Jun;139(6):775-81.
(44) Vischjager M, van Gulik TM, Kromhout JG, van MJ, Pfaffendorf M, Klopper PJ, et al. Morphology and
function of preserved microvascular arterial grafts: an experimental study in rats. Ann Vasc Surg 1997
May;11(3):284-91.
(45) Crowe DM, Hurley JV, Mitchell GM, Niazi Z, Morrison WA. Long-term studies of cold-stored rabbit
femoral artery and vein autografts. Br J Plast Surg 1998 Jun;51(4):291-9.
(46) Marois Y, Wagner E, Paris E, Roy R, Douville Y, Guidoin R. Comparison of healing in fresh and
preserved arterial allografts in the dog. Ann Vasc Surg 1999 Mar;13(2):130-40.
(47) Zhao XM, Green M, Frazer IH, Hogan P, O’Brien MF. Donor-specific immune response after aortic
valve allografting in the rat. Ann Thorac Surg 1994 May;57(5):1158-63.
(48) Komorowska-Timek E, Zhang F, Shi DY, Lineaweaver WC, Buncke HJ. Effect of cryopreservation on
patency and histological changes of arterial isogeneic and allogeneic grafts in the rat model. Ann
Plast Surg 2002 Oct;49(4):404-9.
4
(49) Wang X, Lin P, Yao Q, Chen C. Development of small-diameter vascular grafts. World J Surg
2007 Apr;31(4):682-9.
(50) Yow KH, Ingram J, Korossis SA, Ingham E, Homer-Vanniasinkam S. Tissue engineering of vascular
conduits. Br J Surg 2006 Jun;93(6):652-61.
(51) Borschel GH, Huang YC, Calve S, Arruda EM, Lynch JB, Dow DE, et al. Tissue engineering of
recellularized small-diameter vascular grafts. Tissue Eng 2005 May;11(5-6):778-86.
(52) Gomes D, Louedec L, Plissonnier D, Dauge MC, Henin D, Osborne-Pellegrin M, et al. Endoluminal
smooth muscle cell seeding limits intimal hyperplasia. J Vasc Surg 2001 Oct;34(4):707-15.
(53) Dixit P, Hern-Anderson D, Ranieri J, Schmidt CE. Vascular graft endothelialization: comparative
analysis of canine and human endothelial cell migration on natural biomaterials. J Biomed Mater Res
2001 Sep 15;56(4):545-55.
(54) Rezvan A, Allen FD, Lelkes PI. Steady unidirectional laminar flow inhibits monolayer formation by
human and rat microvascular endothelial cells. Endothelium 2004 Jan;11(1):11-6.
(55) Davids L, Dower T, Zilla P. The lack of healing in conventional vascular grafts. In: Zilla PP, Greisler HP,
editors. Tissue engineering of vascular prosthetic grafts. Austin, Texas: R.G. Landes; 1999, p. 3-44.
Glycerol preserved arterial allografts evaluated in the infrarenal rat aorta | 75
Fahner.indd 75
5-5-2014 14:35:12