Additional Methodology
1. Flow Chart of Study Design
In total 17 animals (41 vessels) were implanted with overlapping Absorb or overlapping XV. 8 and
9 pigs underwent follow up at 28 days (Absorb: n=12, XV: n=8), and 90 days (Absorb: n=12, XV:
n=9) respectively (Appendix).
Figure 1
Title: Flow chart of study design.
Caption: Study pigs implanted with overlapping Absorb or Xience V evaluated by OCT, histology
and scanning electron microscopy, are shown.
Absorb
(n=11)
7 PIGS
(n=18)
8 pigs
28 days
XV
(n=7)
Absorb
(n=11)
17 PIGS
8 PIGS (n=19)
90 days
XV
(n=8)
28 Days
2 PIGS (SEM)*
OCT† (n=11,
413 sections)
HISTO (n=11,
44 sections)
OCT† (n=7,
272 sections)
HISTO (n=7,
28 sections)
OCT† (n=11,
408 sections)
HISTO (n=11,
44 sections)
OCT† (n=8,
314 sections)
HISTO (n=8,
32 sections)
1 Absorb, 1 XV
90 Days
1 Absorb, 1 XV
* 2 separate pigs (from the trial) were scheduled to undergo SEM at 28 and 90 days, consequently no OCT or histology
was performed.
† number of OCT sections analyzed at baseline and follow-up
n (in parentheses) indicates number of vessels with implanted overlapping Absorb or overlapping XV.
Abbreviations: SEM scanning electron microscopy, XV Xience V, OCT optical coherence tomography, HISTO
histology.
2. Absorb and Xience V Devices
The Absorb is a balloon expandable bioresorbable device constructed of poly-L lactide and coated
with a bioresorbable poly-D, L lactide, that contains and controls the release of 100 μg/cm2 of the
anti-proliferative drug everolimus (Novartis, Basel, Switzerland). Approximately 80% of the drug is
released within 30 days after implantation, and the remainder of the drug within 4 months. Based on
studies in a porcine model, the Absorb bioresorption process has been shown to commence at 6
months, with the expected loss of structural integrity and potential restoration of vasomotor
function of the treated vessel at 1 year, (1) and resultant completion of the bioresorption process at
2-3 years. (2) At the time of implantation the total thickness of the Absorb polymeric strut is
approximately 156 μm. The next generation Absorb (1.1), currently the subject of ongoing clinical
trials, was investigated in this study. (3)
The Xience V (XV) is a balloon expandable drug eluting stent, composed of a cobalt-chromium
alloy platform, with a strut thickness of 81 μm and a non-erodible 7.6 μm thick coating. The coating
is composed of an acrylic base and a biocompatible fluorinated copolymer, and has an everolimus
release profile identical to that of the Absorb. The total strut thickness (strut + coating) of the XV is
88.6 μm.(4)
3. Detailed OCT Analyses
OCT evaluation of the Absorb and XV overlap (all) and non-overlap (as long as technically
possible) segments were performed at a pullback speed of 1.0 mm/sec at baseline and at follow-up,
utilizing a commercially available time-domain OCT system (M3 System, LightLab Imaging,
Westford, Massachusetts). The image wire was passed distal to the treated vessel without the
conventional support of the balloon occlusion catheter to minimize the risk of disrupting the
endothelial coverage in the treated vessel. Bolus doses of Ringer's lactate were used to clear blood
distal to the inflated occlusion balloon in the proximal vessel and OCT pullbacks initiated.
Quantitative and qualitative analyses were performed with proprietary software for off-line analysis
(LightLab Imaging, Westford, MA, USA). OCT analyses were performed by a team of physicians
and analysts of an independent core laboratory (Cardialysis BV, The Netherlands), blinded and
independent to the team undertaking the animal and histology analyses (CVPath Institute,
Gaithersburg). With adjustment for pullback speed, analyses of continuous cross-sections were
performed at 1-mm longitudinal intervals consistent with previously validated methodologies,
(3,5,6) and currently being undertaken in ongoing Absorb OCT sub-studies.
Quantitative assessments of the lumen area have been shown to be potentially underestimated with
OCT acquisitions undertaken with an occlusion balloon technique, secondary to the nonphysiological pressurization of the coronary vessel. (7) Since all lumen measurements were
undertaken in coronary segments scaffolded either by a metallic stent, or polymeric scaffold that
maintained its mechanical integrity at 90 days, these effects would have been minimized.
The main quantitative measurements (strut area, lumen area, scaffold area, % volume obstruction
and neointimal area) and OCT end points related to the interaction of the struts and vessel wall (e.g.
apposition and coverage) for the Absorb (3,8) require different analysis rules compared to
conventional stents (XV), (5,9) and have been described previously. Specifically for the
overlapping Absorb, struts were defined by their overlay configuration, namely ‘stacked inner’,
‘stacked outer’ and ‘other’ (i.e. without a clear direct overlay configuration) (Fig. 1). (8) Given the
heterogeneity in the neointimal coverage dependent on the overlay configuration of the Absorb
struts, neointimal areas are reported to allow appropriate comparisons between the overlap and nonoverlap segments and between devices.
Strut coverage of the Absorb was assessed as a thickness coverage measured between the mid part
of the luminal edge of the strut, following the center of gravity to the lumen boundary, for all strut
types at 28 and 90 days (Fig. 2). The threshold for coverage of the Absorb strut is 30 μm,
corresponding to the average interobserver measurement (difference in 300 struts analyzed two
times, 35±6 μm) of the endoluminal light backscattering strut boundary. (10) All reviewers were
blinded to this cut-off value, which was implemented by an independent statistician during data
analysis.
To allow full visualization of the spatial distribution of strut coverage struts in the overlapping
devices, ‘spread-out-vessel graphs’ – a visual representation of the vessel as if it had been cut along
the reference angle (0°) and spread out on a flat surface – were created based upon previously
described methodologies. (5)
Figure 1
Title: Examples of Absorb struts according to their overlay configuration in the overlap.
Caption: ‘Stacked inner’, ‘stacked outer’ and ‘other’ struts are illustrated on OCT (left) and
scanning electron microscopy (right). White and grey arrows indicate ‘stacked inner’ struts with
corresponding ‘stacked outer’ struts located abluminally – defined as struts with a direct overlay
configuration with each other or lying within 1 strut width of each other. Blue arrows indicates
‘other struts’ i.e. struts in the overlap without a clear direct overlay configuration. Grey arrow
indicates a strut attached to a platinum marker used to visualize the Absorb during implantation.
Broken white lines illustrate selected examples of Absorb struts where the neointimal coverage is
measured from (mid part of the luminal side of ‘black core’ area of the Absorb strut) to the lumen
boundary.
4. Histological Analyses and Scanning Electron Microscopy (SEM)
Following euthanasia, the hearts were explanted from the thoracic cavity and flushed using pressure
perfusion with saline followed by 10% buffered formalin. Implanted arteries were carefully
dissected from the heart, routinely processed, embedded in methyl methacrylate, and sectioned in
duplicate at 5 µm according to published procedures. (11) Two to three millimeter sections were
sawn in 4 segments, from the proximal non-overlap (1 section), mid overlap (1 proximal and 1
distal overlap section) and distal non-overlap (1 section). Sections were stained with hematoxylin
and eosin and elastic van Gieson or Movat pentachrome. The practice of undertaking in vivo OCT
and ex vivo histology precluded the precise correlation of individual cross sections and matched
analyses, since no external marker could be implanted to act as a point of reference. Standard
histomorphometric assessments, vascular injury, fibrin deposition and inflammatory responses in
accordance with established scoring systems were evaluated. (11,12) Strut coverage was assessed as
the % of struts with endothelial cell and/or cellular coverage. Endothelial coverage was semiquantified and expressed as a % of the lumen circumference covered by endothelium. These 2
assessments were separately undertaken because of the risk of endothelial denudation due to the
OCT procedure performed immediately prior to animal sacrifice. (13) In the 2 pigs designated for
SEM, the arterial segments were prepared and visualization undertaken with a Hitachi Model
3600N scanning electron microscope as previously described. (14) The luminal surface was
qualitatively assessed.
Additional Results
1. Histological and SEM Findings
Table 1 provides the histomorphometric and qualitative histomorphologic findings in the overlap
and non-overlap. At all time points the injury scores were comparable between the overlap and nonoverlap and between devices. For the overlapping Absorb and XV devices, the mean inflammation
scores were low and comparable at 28 and 90 days. Mean fibrin scores were similarly low and
followed a trend of regression from 28 to 90 days, consistent with the elution profile of everolimus
from both devices. (4) The % endothelialization of the overlapping Absorb appeared reduced at 28
(p=0.018) and 90 days (p=0.069) compared to the overlapping XV.
Table 1: Results of the qualitative histomorphometric and histological data at the time points for the
2 separate animal groups at 28 and 90 days. Data presented as means ± SD.
28 Days
EEL area (mm2)
IEL area (mm2)
Medial area (mm2)
Lumen area (mm2)
Neointima area (mm2)
% volume obstruction
Neointimal Thickness
Injury score†
Mean fibrin score
Endothelialization (%)
Covered struts (%)
Inflammation score
Adventitial inflammation score
90 Days
EEL area (mm2)
IEL area (mm2)
Medial area (mm2)
Lumen area (mm2)
Neointima area (mm2)
% volume obstruction
Neointimal Thickness
Injury score†
Mean fibrin score
Endothelialization (%)
Covered struts (%)
Inflammation score
Adventitial inflammation score
Overlap
Absorb
Non
Overlap
pvalue
Overlap
XV
Non
Overlap
p-value
Overlap
p-value*
9.89±1.02
8.43±0.80
1.46±0.45
5.75±0.71
2.67±0.31
31.84±3.43
0.032±0.024
0.38±0.15
2.00±0.63
67.95±14.87
75.4±16.4
1.00±0.00
0.091±0.20
8.95±0.77
7.60±0.63
1.35±0.22
5.81±0.57
1.80±0.29
23.71±3.67
0.080±0.034
0.32±0.23
1.50±0.39
82.18±7.99
88.8±24.5
0.41±0.38
0.00±0.00
0.004
0.002
0.26
0.92
<0.001
<0.001
<0.001
0.48
0.023
<0.001
<0.001
<0.001
0.13
8.71±1.30
7.53±1.06
1.17±0.25
5.52±1.09
2.01±0.36
27.15±6.56
0.12±0.040
0.45±0.19
1.79±0.70
87.43±15.92
99.6±1.46
0.71±0.91
0.21±0.57
7.80±1.28
6.67±1.09
1.13±0.24
5.37±1.08
1.30±0.15
20.06±3.60
0.12±0.031
0.49±0.31
1.07±0.53
93.14±8.23
100.0±0
0.57±0.93
0.29±0.57
0.14
0.12
0.72
0.84
<0.001
0.008
0.89
0.69
0.029
0.21
0.76
0.43
0.98
0.047
0.057
0.15
0.59
<0.001
0.063
<0.001
0.34
0.42
0.018
<0.001
0.058
0.94
10.60±2.01
8.39±0.56
2.21±2.12
4.41±1.34
3.98±1.04
47.90±14.37
0.25±0.18
0.71±0.69
0.68±0.51
63.18±29.37
98.7±3.7
0.59±1.22
0.55±1.21
9.29±1.48
7.51±0.59
1.78±1.33
4.96±1.25
2.55±1.07
34.20±14.52
0.18±0.18
0.81±0.69
0.27±0.47
58.91±27.75
100±0
0.64±1.16
0.64±1.19
0.005
<0.001
0.33
0.17
<0.001
0.003
0.025
0.18
0.014
0.65
0.076
0.63
0.80
8.83±1.33
7.71±1.20
1.13±0.15
4.90±1.87
2.81±1.16
37.78±16.97
0.24±0.14
0.76±0.76
0.31±0.46
85.13±14.27
100±0
0.75±1.36
0.69±1.28
7.96±1.18
6.87±1.08
1.08±0.14
5.04±1.44
1.83±0.55
27.84±10.73
0.18±0.083
0.69±0.63
0.19±0.37
70.44±24.24
99.0±2.9
0.69±1.36
0.25±0.53
0.063
0.054
0.48
0.81
0.005
0.059
0.15
0.87
0.56
0.070
0.56
0.96
0.62
0.046
0.11
0.17
0.51
0.034
0.18
0.88
0.87
0.12
0.069
0.49
0.61
0.82
* P-value comparing overlapping Absorb vs. overlapping XV
† Mean injury score per overlap or non-overlap segment. Overall these injury scores are low and are within expected
limits for a study of this design.
Abbreviations: µm micrometer, XV Xience V, % percentage, mm millimeter, SD standard deviation
Additional Discussion
1. Comparison of overlapping Absorb and XV devices
Despite comparable injury scores in vessels treated with either device, the overlapping Absorb
exhibited a greater neointimal response in absolute terms compared to the non-overlapping Absorb
segments. This however did not translate into a significantly greater relative % volume obstruction
between either device on OCT analyses, due to a greater vessel/scaffold area associated with the
Absorb overlap.
Conversely, histological analyses suggested a greater % volume obstruction with overlapping
Absorb compared to overlapping XV at 28 (p=0.063) and 90 days (p= 0.18). Explanations for this
discrepancy include that during histological preparation, a metallic stent would less likely shrink
compared to the Absorb, and that formalin fixation and dehydration would result in vessel
shrinkage despite the vessel pressure being arbitrarily fixed at 100 mg. Comparatively OCT was
performed in vivo, where the coronary vessel had tonus and was naturally pressurized, with the
resultant increased distension of the vessel. (15)
As previously stated, the significantly greater scaffold area by OCT and EEL area by histology, of
the overlapping Absorb (compared to the non-overlap segments) allowed for the accommodation of
the increased neointima area associated with the over-lapping Absorb. Importantly, OCT and
histology analyses (independently performed in different core laboratories) corroborated each
other’s findings. As to why the overlapping Absorb had a greater scaffold/EEL area, it is likely that
the addition of overlapping Absorb struts on either side of the deploying device balloon increased
the vessel size by 4 layers of Absorb struts (>600 µm thickness), without rupturing the IEL and
vessel media. In addition, the more conformable, thicker Absorb struts (compared to metal) (16)
may have reduced its ‘cutting effect’ when embedded in the vessel media, without any resultant
increase in vessel injury. Previous pre-clinical studies have however shown that over-stretching of
the vessel can induce mechanical injury to neointimal SMCs, leading to changes in synthetic SMC
phenotypes, with a resultant increase in constrictive remodeling. (17) The possibility of a greater
neointimal response associated with high grade stenotic lesions treated with overlapping Absorb
can therefore not be excluded, as previously reported in one clinical study. (18)
2. Endothelial Coverage
Notably the endothelial coverage of the overlapping Absorb appeared reduced at 28 (p=0.018) and
90 (p=0.069) days compared to the overlapping XV. Paradoxically, almost complete cellular
coverage was achieved in either overlapping device at 90 days, with low and comparable fibrin and
inflammation scores. It has previously been shown that coronary wire manipulation and/or vascular
imaging with intravascular ultrasound or OCT can induce acute endothelial damage (up to a level of
38% reported by SEM in a previous porcine study (13)) which heals within 5 days. Consequently
the clinical significance of the reduced endothelialization by histology in the present study is
unclear, particularly since both overlapping Absorb and XV cases on SEM at 90 days demonstrated
that re-endothelialization was fully complete.
3. Clinical implications of bioresorption of the Absorb in the overlap
The main clinical question is therefore at what stage will full neointimal coverage be achieved in
vessels implanted with overlapping Absorb? Since the polymeric struts are associated with a long
bioresorption process (2-3 years), the classic correlation between peak neointimal growth in the
porcine model and humans in standard metallic stents may not be valid. Porcine studies have
however shown no further increase in the neointimal volume from 90 days to 1 year in singly
implanted Absorb (unpublished data). Furthermore the possibility of an adaptive expansive
remodeling process, with the prospect of ‘late luminal enlargement,’ may occur as early as one year
following partial bioresorption and expected loss of structural integrity of the Absorb device, and
may prove to be of additional clinical value. (1,19)
The Absorb is one of a number of bioresorbable devices that are either in pre-clinical or clinical
testing. (20,21) One inherent feature in all bioresorbable scaffolds – and notably first generation
DES – and not unique to the Absorb device, is the increased strut thickness compared to second
generation metallic DES. The increased strut thickness of bioresorbable scaffolds is to allow for
adequate radial strength of a sufficient period of time to prevent early recoil, and adequate
distensibility of the device to permit safe implantation. (22) As an example, the second generation
bioresorbable ‘DREAMS’ (the Drug Eluting Absorbable Metal Scaffold) device is now undergoing
clinical testing, and has a reported strut thickness of approximately 150 µm. (20) Current and
future bioresorbable devices may therefore require design features to allow for minimal overlap or
adjacent positioning of the devices, (8) longer devices, and possibly dedicated bifurcations devices
to eliminate the need to overlap the devices during complex 2 stent coronary bifurcation procedures.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Brugaletta S, Heo JH, Garcia-Garcia HM et al. Endothelial-dependent vasomotion in a
coronary segment treated by ABSORB everolimus-eluting bioresorbable vascular
scaffold system is related to plaque composition at the time of bioresorption of the
polymer: indirect finding of vascular reparative therapy? European Heart Journal 2012
2012;33:1325-33.
Onuma Y, Serruys PW, Perkins LE et al. Intracoronary optical coherence tomography
and histology at 1 month and 2, 3, and 4 years after implantation of everolimus-eluting
bioresorbable vascular scaffolds in a porcine coronary artery model: an attempt to
decipher the human optical coherence tomography images in the ABSORB trial.
Circulation 2010;122:2288-300.
Serruys PW, Onuma Y, Ormiston JA et al. Evaluation of the second generation of a
bioresorbable everolimus drug-eluting vascular scaffold for treatment of de novo
coronary artery stenosis: six-month clinical and imaging outcomes. Circulation
2010;122:2301-12.
Gomez-Lara J, Brugaletta S, Farooq V et al. Head-to-head comparison of the neointimal
response between metallic and bioresorbable everolimus-eluting scaffolds using
optical coherence tomography. JACC Cardiovascular interventions 2011;4:1271-80.
Gutierrez-Chico JL, van Geuns RJ, Regar E et al. Tissue coverage of a hydrophilic
polymer-coated zotarolimus-eluting stent vs. a fluoropolymer-coated everolimuseluting stent at 13-month follow-up: an optical coherence tomography substudy from
the RESOLUTE All Comers trial. European Heart Journal 2011;32:2454-63.
Gutierrez-Chico JL, Regar E, Nuesch E et al. Delayed coverage in malapposed and sidebranch struts with respect to well-apposed struts in drug-eluting stents: in vivo
assessment with optical coherence tomography. Circulation 2011;124:612-23.
Gonzalo N, Serruys PW, Garcia-Garcia HM et al. Quantitative ex vivo and in vivo
comparison of lumen dimensions measured by optical coherence tomography and
intravascular ultrasound in human coronary arteries. Revista Espanola de Cardiologia
2009;62:615-24.
Farooq V, Onuma Y, Radu M et al. Optical coherence tomography (OCT) of overlapping
bioresorbable scaffolds: from benchwork to clinical application. EuroIntervention:
2011;7:386-99.
Guagliumi G, Musumeci G, Sirbu V et al. Optical coherence tomography assessment of
in vivo vascular response after implantation of overlapping bare-metal and drugeluting stents. JACC Cardiovascular Interventions 2010;3:531-9.
Serruys PW, Onuma Y, Dudek D et al. Evaluation of the second generation of a
bioresorbable everolimus-eluting vascular scaffold for the treatment of de novo
coronary artery stenosis: 12-month clinical and imaging outcomes. 2011;58:1578-88.
Perkins LEL, Boeke-Purkis KH, Wang Q, Stringer SK, Coleman LA. XIENCE V™
Everolimus-Eluting Coronary Stent System: A Preclinical Assessment. Journal of
Interventional Cardiology 2009;22:S28-S40.
Schwartz RS, Huber KC, Murphy JG et al. Restenosis and the proportional neointimal
response to coronary artery injury: results in a porcine model. J Am Coll Cardiol
1992;19:267-74.
Abstract van Beusekom H, Van Duin R, Krabbendam-Peters I, Van Haeren R, van der
Giessen WJ Single and repeated endovascular imaging cause significant but equal acute
endothelial injury of a temporary nature as opposed to stent induced injury. Presented
EuroPCR 2011, Paris, France Published in EuroIntevention Volume 7 Supplement M on
14.
15.
16.
17.
18.
19.
20.
21.
22.
May 17, 2011. http://wwwpcronlinecom/eurointervention/M_issue/220. Accessed
25th May 2012.
Sheehy A, Hsu S, Sinn I et al. Vascular response to coronary artery stenting in mature
and juvenile swine. Cardiovascular Revascularization Medicine: including molecular
interventions 2011;12:375-84.
Gogas BD, Radu M, Onuma Y et al. Evaluation with in vivo optical coherence
tomography and histology of the vascular effects of the everolimus-eluting
bioresorbable vascular scaffold at two years following implantation in a healthy
porcine coronary artery model: implications of pilot results for future pre-clinical
studies. The International Journal of Cardiovascular Imaging 2012 Mar;28(3):499-511.
Gomez-Lara J, Brugaletta S, Farooq V et al. Angiographic geometric changes of the
lumen arterial wall after bioresorbable vascular scaffolds and metallic platform stents
at 1-year follow-up. JACC Cardiovascular Interventions 2011;4:789-99.
Liu MW, Roubin GS, King SB, 3rd. Restenosis after coronary angioplasty. Potential
biologic determinants and role of intimal hyperplasia. Circulation 1989;79:1374-87.
Tahara S, Bezerra HG, Sirbu V et al. Angiographic, IVUS and OCT evaluation of the longterm impact of coronary disease severity at the site of overlapping drug-eluting and
bare metal stents: a substudy of the ODESSA trial. Heart 2010;96:1574-8.
Serruys PW, Garcia-Garcia HM, Onuma Y. From metallic cages to transient
bioresorbable scaffolds: change in paradigm of coronary revascularization in the
upcoming decade? European Heart Journal 2012;33:16-25b.
Waksman R. The disappearing stent: when plastic replaces metal. Circulation
2012;125:2291-4.
Onuma Y, Serruys PW. Bioresorbable scaffold: the advent of a new era in percutaneous
coronary and peripheral revascularization? Circulation 2011;123:779-97.
Farooq V, Gomez-Lara J, Brugaletta S et al. Proximal and distal maximal luminal
diameters as a guide to appropriate deployment of the ABSORB everolimus-eluting
bioresorbable vascular scaffold. Catheterization and Cardiovascular Interventions
2012;79:880-8.