ARTICLE ORIGINAL
Preliminary evaluation of the biocompatibility
of the small intestinal submucosa (SIS)
biomaterial with the rabbit cornea
° F. GRIGUER, °° I. RAYMOND and ° A. REGNIER
° Département des Sciences Cliniques, École Nationale Vétérinaire, 23 chemin des Capelles, F-31076 Toulouse Cedex 03
°° Département des Sciences Biologiques et Fonctionnelles, École Nationale Vétérinaire, 23 chemin des Capelles, F-31076 Toulouse Cedex 03
Correspondence to : A. Regnier, Clinique d’Ophtalmologie, École Nationale Vétérinaire, 23 chemin des Capelles, F-31076 Toulouse Cedex 03
E-mail : a.regnier@envt.fr
SUMMARY
The early stages of the ocular response to porcine small intestinal sub-
mucosa (SIS) transplantation were assessed in a model of anterior keratec-
tomy in the rabbit. After surgery, the operated eyes (n = 17) were examined
on a regular basis over a 15-day period and animals were sacrified at pre-
determined times for histopathologic evaluation. In all but one animal, the
biomaterial was well tolerated, and the corneal response was restricted to a
localized neovascularization. The grafted material was epithelialised within
7 days and incorporated into the corneal tissue without inducing granuloma
formation. In one rabbit, the corneal inflammation was severe but the bio-
material was maintained in place. Although further studies with longer fol-
low-up periods are needed, these preliminary observations suggest that por-
cine SIS can be incorporated into the corneal tissue during healing process
and may represent a promising graft material for lamellar keratoplasty.
KEY-WORDS : small intestinal submucosa - corneal tole-
rance - lamellar keratoplasty - rabbit.
Introduction
The concept of a resorbable matrix replaced by autologous
tissue is currently gaining acceptance as an effective proce-
dure for tissue reconstruction in numerous organ systems.
This technique involves the use of a biodegradable scaffold
that the host tissue can use to remodel and regenerate. One
particular degradable biomaterial, small intestinal submu-
cosa (SIS), has stimulated research investigation and clinical
interest in the last few years. SIS is derived from the submu-
cosal layer of the porcine jejunum and consists of the stratum
compactum layer of the tunica mucosa, the tunica muscularis
mucosa, and the tunica submucosa [5]. This biomaterial is
Revue Méd. Vét., 2001, 152, 8-9, 597-604
RÉSUMÉ
Étude préliminaire de la biocompatibilité d’une greffe lamellaire de
sous-muqueuse intestinale dans la cornée du lapin. Par F. GRIGUER,
I. RAYMOND et A. REGNIER.
La réaction oculaire à une greffe cornéenne lamellaire de sous-muqueu-
se intestinale de porc (small intestinal submucosa : SIS) a été étudiée sur un
modèle de kératectomie superficielle chez le lapin. Après la chirurgie, un
suivi clinique des yeux opérés (n = 17) a été fait régulièrement pendant
15 jours et les animaux ont été sacrifiés à des temps prédéterminés afin de
réaliser une étude histopathologique des globes oculaires. Les résultats cli-
niques et histopathologiques montrent qu’à l’exception d’un cas, la greffe
lamellaire de SIS a été bien tolérée et n’a entraîné qu’une néovascularisa-
tion localisée. Le greffon de SIS a été épithélialisé en 7 jours et il s’est inté-
gré dans le stroma receveur sans entraîner de réaction inflammatoire de type
corps étranger. Dans un cas, la réaction inflammatoire de la cornée a été
marquée mais le greffon n’a pas été rejeté. Bien que des études à plus long
terme soient nécessaires, ces résultats préliminaires suggèrent que la sous-
muqueuse intestinale est un biomatériau qui peut s’intégrer au processus de
cicatrisation de la cornée et pourrait en cela servir pour la kératoplastie
lamellaire.
MOTS-CLÉS : sous-muqueuse intestinale - tolérance
cornéenne - kératoplastie lamellaire - lapin.
primarily an acellular extracellular matrix composed of col-
lagen, proteoglycans, glycoproteins, and glycosaminogly-
cans [8]. It also contains natural growth factors including
basic fibroblast growth factor (basic FGF) and transforming
growth factor beta (TGF-beta) [31].
SIS has several interesting physical and biological proper-
ties : it has good tensile properties [33], is nonimmunogenic
[5], is resistant to infection [6], and when used as a xenograft
material promotes wound healing by providing a scaffold for
tissue ingrowth [24]. As a result, the collagen matrix is degra-
ded and is replaced by an autologous tissue that closely
resembles the native structure [5, 11].
598
Since 1989, xenogeneic porcine SIS has been successfully
used in laboratory animals for arterial or venous replacement
[4, 6] and reconstruction of urinary bladder [21 22], cranial
dura mater [12], meniscus [13], abdominal wall [11] or
Achilles tendon [7]. Porcine SIS has recently become avai-
lable on the veterinary market and is currently used as a bio-
logical dressing for skin wounds in small animals and horses.
Although recent reports disclosed encouraging results with
the use of porcine SIS for tectonic keratoplasty in one dog
[26] and a small series of cats [15], the fundamental question
of SIS biocompatibility with the cornea remains unanswered.
In an attempt to address this question, a study was designed
to evaluate the early phase of the ocular response to a lamel-
lar grafting of SIS in a rabbit model of keratectomy wound.
Results from this study were reported as a clinical research
abstract at the British Small Animal Veterinary Association
Congress in Birmingham, 6-9 April 2000.
Materials and methods
TEST MATERIAL
Under the lamellar flow hood, the grafts were prepared
from a sterile sheet of SIS (7.0 cm x 4.0 cm) available for
veterinary surgery (Vet Biosist®, Cook Veterinary Products).
The dehydrated sheet was divided into discs, measuring
7.0 mm in diameter, with a corneal trephine. The discs were
placed in sterile individual vials containing a sterile solution
for intraocular irrigation (Aqsia®, Chauvin Opsia SA) and
gentamicin at a concentration of 100 µg/ml (Gentalline®
injectable 40 mg, Schering-Plough). The vials with the
enclosed disc of SIS were stored at 4°C until use.
ANIMALS AND SURGICAL TECHNIQUE
Seventeen healthy male New Zealand White rabbits
without ocular disease and weighing 2.5 to 3 kg were used in
accordance with the ARVO Resolution on the Use of
Animals in Research. They were kept in individual cages
under standard animal room conditions, fed dry pellets and
had water ad libitum. All rabbits were anesthetized intramus-
cularly with 50 mg/kg body weight ketamine hydrochloride
(Kétamine 1000®, Virbac Animaux de Compagnie) and
5 mg/kg body weight xylazine hydrochloride (Rompun®,
Bayer Pharma). To supplement the general anesthesia, topi-
cal 0.4 % oxybuprocaine (Novésine®, MSD-Chibret) was
applied to the eye selected for grafting.
The eye to be operated was selected at random for each
rabbit. A pediatric eyelid speculum was used to expose the
globe and stay sutures of 4/0 silk were placed at 6 and 12
o’clock to stabilize the globe. Each animal underwent a cen-
tral lamellar keratectomy. A 7.0 mm corneal trephine with
depth adjustment to 0.2 mm was used to standardize the inci-
sion. The circumscribed epithelium and stroma were remo-
ved with a Peaufique corneal dissector. A disc of SIS, similar
in size to the keratectomy, was removed from the storage
medium and placed over the corneal defect with its serosal
side (tunica submucosa) facing the recipient bed. The graft
was secured to the edge of the corneal defect with 12 simple
GRIGUER (F.) AND COLLABORATORS
interrupted 10/0 nylon sutures. Chloramphenicol ophthalmic
ointment (Ophtalon®, TVM) was applied on the operated
eye immediately following grafting and twice daily for
7 days after surgery.
CLINICAL FOLLOW UP
The recipient eyes were examined by slitlamp biomicro-
scopy on postoperative days 1, 2, 3, 4, 5, 6, 7, 10 and 15.
According to their severity, conjunctival injection and che-
mosis, oedema of the peripheral cornea and iritis, characteri-
zed by miosis and iridial hyperemia, were scored as follows : 0
= absent ; 1 = mild ; 2 = moderate, and 3 = severe. The extent
of corneal vascularization was assessed on the basis of the
farthest vascular ingrowth from the limbus as follows :
0 = no corneal vascularization ; 1 = 2-mm vascular ingrowth ;
2 = 4-mm vascular ingrowth, and 3 = 6-mm vascular
ingrowth. Biomicroscopic examination also evaluated the
integrity and appearance of the grafted material.
HISTOPATHOLOGIC STUDIES
The animals were sacrified with an intravenous overdose
of barbiturate at 2 (n = 2), 4 (n = 2), 7 (n = 4), 10 (n = 3), and
15 days (n = 6) postoperatively. After enucleation, the opera-
ted eyes were fixed in 10 % neutral buffered formaldehyde.
The corneas with a rim of sclera were dissected from the fixa-
ted globes and mounted in paraffin. Sections 6 µm thick were
stained with haematoxylin and eosin (H & E), Masson’s tri-
chrome, and periodic acid Schiff (PAS) stains to be examina-
ted by light microscopy.
Results
SURGICAL TECHNIQUE
In all cases, surgery was uneventful and technically not dif-
ficult because the SIS was tough and thick enough to hold the
100 sutures without tearing.
GROSS AND BIOMICROSCOPIC FINDINGS
Postoperatively, none of the 17 rabbits showed evidence of
discomfort and photophobia. Consistent degrees of conjunc-
tival injection and chemosis were evident in all operated eyes
in the early postoperative period. The conjunctival injection
and chemosis reached a maximum at day 1, with a mean
score of 1.65 and 1.35 respectively, and then gradually dimi-
nished to disappear by days 10-15 (Fig. 1). In all but one ani-
mal (rabbit 12), no corneal oedema was observed and the cor-
nea around the SIS graft remained transparent throughout the
follow-up period. There was no evidence of stromal melting
around the keratectomy area in any of the operated eyes for
the 15 days of the experimental period. In 11 of 13 (approxi-
mately 85 %) of the cases that were followed for at least
7 days, vessels could be seen invading the peripheral cornea
from 4 to 6 days after surgery (Fig. 1). Vascular sprouts arose
from the circumlimbal vessels and formed a small band,
approximately 5 mm wide, in the superotemporal quadrant.
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PRELIMINARY EVALUATION OF THE BIOCOMPATIBILITY OF THE SMALL INTESTINAL SUBMUCOSA (SIS)
FIGURE 1. — Effect of corneal implantation of SIS on individual parameters
of the ocular response in rabbits. Each point represents mean score for
parameter on designated day of postoperative follow-up (see text for sco-
ring procedures). Vertical lines indicate 1 SD.
The neovessels were superficial and few in number. They
progressively grew, perpendicular to the limbus and toward
the region of the graft, to a length of 2-4 mm which persisted
throughout the experiment. Moderate signs of iritis, manifes-
ted as iris vascular injection and/or miosis, were present in all
eyes for the first 4-6 postoperative days (maximum mean
score = 1.53) and subsequently regressed without sequela
(Fig. 1).
During the first 48 hours following surgery, there was no
visible change in the appearance of the grafts (Fig. 2A). From
postoperative days 3-4 to 6-10, the xenografts appeared opa-
lescent and swollen (Fig. 2B). Their superficial layers were
deliquescent and sloughed off, forming small whitish debris
at the surface of the grafted area (Fig. 2C). Nevertheless, all
grafts were retained on the wound bed and became progressi-
vely less opaque by postoperative days 8-10. In parallel, their
surface became smoother and brighter. By postoperative day
Revue Méd. Vét., 2001, 152, 8-9, 597-604
599
15, the grafts were partly resolved and the grafted areas were
sufficiently transparent to see the iris and pupil (Fig. 2D).
In one animal (rabbit 12), a severe corneal oedema (clinical
score = 3) with neovessels invading the stroma and a marked
uveitis (clinical score = 3) were observed 2 days after sur-
gery. Uveitis improved by approximately 10 days and left no
visible signs. On completion of the experiment, the cornea
was markedly vascularized (vessels reached 6 mm in length)
and slightly oedematous. The grafted area was opaque and
partly covered with fibrovascular tissue (Fig. 3).
HISTOPATHOLOGIC FINDINGS
On the 2nd and 4th postoperative days, a discrete superfi-
cial infiltration of the stroma consisting of polymorphonu-
clear (PMN) cells and scarce mononuclear cells occurred
around the wound bed. The superficial layers of the graft
appeared as loose collagen sheets coming off from the
remainder of the grafted material. Epithelial cells surroun-
ding the keratectomy area had begun to proliferate and
migrate down the side of the wound and over the external
surface of the graft. Few epithelial cells were also present
between the loose superficial layers of the graft and at the
periphery of the graft-host interface where they appeared as
flat, endothelial-like cells.
Seven days after SIS implantation, the stromal inflamma-
tory infiltration had almost disappeared in the middle of the
wound and decreased at its periphery. The superficial layers
of the graft had been expelled, and the anterior surface of the
remaining material was covered with 1 to 2 layers of squa-
mous-like epithelial cells (Fig. 4). By this time, young and
activated fibroblasts were observed in the stroma close to the
graft-host interface and islets of newly grown epithelium
were sometimes found entrapped within the graft (Fig. 4).
Thin and superficial vessels extending from the dorsal limbus
toward the corneal apex were identified.
In all but one (rabbit 12) specimens obtained on the 10th
and 15th postoperative days, a mild residual inflammatory
cellular infiltrate was present around the suture sites. At
10 days, the grafts were totally covered with an epithelium
composed of one to two layers of squamous cells and a single
layer of basal cells that were cuboidal and lightly stained
(Fig. 5). Activated keratocytes were present in great number
just under the grafted material and were identified between
the collagen fibres of the graft remnants (Fig. 5). By 15 days
post-grafting, PAS staining sections showed that a new base-
ment membrane was present under the epithelium (Fig. 6). At
that time, the corneal thickness was restored and the newly
formed collagen fibres appeared more regularly oriented than
those of the graft remnants and grossly parallel to the corneal
surface (Fig. 7). Histology of the cornea from rabbit 12 sho-
wed a severe infiltration of the grafted material and surroun-
ding stroma by PMNs and a few mononuclear cells.
Remnants of the SIS appeared fragmented and disorganised,
and epithelial coverage of the graft was incomplete (Fig. 8).
At each time point of the follow-up, the other structures of
the anterior and posterior segments appeared to be histologi-
cally normal.
600
Discussion
Deep ulcers are a common problem in small animal oph-
thalmology and require prompt diagnosis and adequate treat-
ment. In addition to medical therapy a number of surgical
options can be used, each having both advantages and limita-
tions. Conjunctival grafts are probably the most commonly
used surgical techniques. Although these procedures have
been shown to be effective, they can also lead to scarring and
pigmentation of the recipient cornea [18]. Corneoscleral
transposition, autologous sliding lamellar keratoplasty, or
homologous lamellar corneal transplantation is also useful
when there is a risk of perforation, as each of these tech-
niques is aimed to replace the damaged stroma and promote
healing with re-epithelialisation [16, 35]. These three tech-
niques have the advantages to allow for better cosmetic
appearance and functional vision than conjunctival grafts
[16]. As the use of fresh or preserved homologous lamellar
grafts may be limited by the difficulty of harvesting and/or
storing donor tissue preparation [17], some investigators
have evaluated the possibility of repairing corneal ulcers with
various biological membranes that are more readily available
than homologous corneal grafts. For instance, preserved
homologous amniotic membrane has been used in humans to
treat deep corneal ulcers [25], and transplantation of preser-
ved xenologous pericardium [2], renal capsule [1] or amnio-
tic membrane [3] was a successful treatment of experimental
corneal defects in dogs. Despite these results, major limita-
tions for clinical application of these techniques appear to be
the preparative and storage procedures to which the trans-
plants should be submitted before use [25]. Such drawbacks
might be eliminated by using a biomaterial ready to use, such
as the SIS marketed for veterinary surgical applications.
In the present study, the lamellar grafting of SIS was found
to produce a mild-to-moderate degree of inflammatory
adverse reactions, both clinical and histopathologic.
Transient conjunctival inflammation and iritis were consis-
tently observed in the early postoperative phase. This non
specific response was likely due to the trauma of surgery
since it is well known that the rabbit eye has a high sensitivity
to the surgical trauma and rapidly responds by an inflamma-
tory reaction, including conjunctival hyperemia, chemosis,
iridial hyperemia, and miosis [10, 34]. With the exception of
one case, no obvious symptoms of graft rejection were obser-
ved since the corneas remained clear throughout the follow-
up period and had a developing reaction to SIS that was
confined to a moderate neovascularization. Neovessels were
produced from a small area of the peripheral cornea and were
apparent in 3 to 6 days. This rate of vascularization coincides
with other reported observations on corneal angiogenesis
induced by xenografts in rats [9] and biodegradable polymers
in rabbits [19]. Histologically, a slight degree of PMN infil-
tration was observed around the keratectomy area in the early
postoperative days. There was no evidence of granuloma for-
mation, a finding which is coherent with previous reports on
body wall [11] and bladder wall response [20, 22] to SIS
implantation. It is likely that there were at least two routes for
the leukocytic infiltration. In the first one, the grafted mate-
rial, although sutured to the cornea, did not provide an effec-
GRIGUER (F.) AND COLLABORATORS
tive barrier against the tear film and consequently did not pre-
vent the influx of PMNs from tears. In the other one, soluble
components within SIS might act as chemoattractants to sti-
mulate the inflammatory reaction. As such, TGF-beta is
known to have chemotactic properties to stimulate corneal
leukocytic infiltration [32] and angiogenesis [27]. In the cur-
rent study, the leukocyte infiltration preceded neovasculari-
zation and initiation of angiogenesis was very likely a conse-
quence of the leukocytic infiltration, since corneal angioge-
nesis is reportedly facilitated by the presence of leukocytes in
the stroma [28]. This response might be amplified by a
variety of growth factors contained in SIS. For instance, it
has been shown that basic FGF can stimulate corneal angio-
genesis without inducing an inflammatory reaction [19].
The ocular reaction following SIS grafting was severe in
only one operated eye (rabbit 12) and overall, many of the
clinical features of this case paralleled those found in corneal
xenograft rejection [9]. Nevertheless, the SIS graft was main-
tained in place, and the ocular inflammation became less
intense 10 days after surgery. Histologically, the components
of the inflammatory infiltrate consisted primarily of PMN
leucocytes with fewer lymphocytes and plasma cells. These
histopathological features are different from those observed
in rejecting corneas, that are characterized by an accumula-
tion of lymphocytes and monocytes [30]. Although current
speculation is that there is no inherent danger of an immuno-
logic reaction, because SIS biomaterial does not contain live
cells [23], our observation raises the question whether SIS
may elaborate an immunogenic response when implanted in
the cornea. Further studies will be needed to elucidate this
hypothesis. Pre-operative SIS biomaterial contamination or
post-operative infection may be other possibilities to explain
the inflammatory reaction in this animal. Although these
hypotheses cannot be dismissed, the risk of bacterial conta-
mination was minimized by adding gentamicin to the storage
solution and applying chloramphenicol ophthalmic ointment
during the first postoperative week.
This study also examined the early phases of corneal epi-
thelium and stromal reconstruction following SIS transplan-
tation, and our preliminary results suggest that this biomate-
rial was incorporated into the cornea during a wound-healing
response. During the first postoperative days, the SIS graft
seems to serve both as a patch and a graft for re-establish-
ment of the epithelial cells because epithelialisation can take
place over, inside and underneath the grafted material.
Although the biomaterial was secured to the keratectomy
wounds with the more densely compacted side (stratum com-
pactum) external, epithelialisation of the graft from neighbo-
ring corneal epithelium was not facilitated in the first post-
operative days since the newly formed epithelium was elimi-
nated with the superficial layers of the graft. In comparison,
a 7 mm keratectomy wound in the rabbit is spontaneously
covered by epithelial cells in approximately 66 hours [37]. In
the current study, the epithelium was reestablished above the
grafted material at day 7 and stratified from days 10 to 15. At
this early stage after grafting, the number of cell layers was
less than in a normal rabbit corneal epithelium but despite
this, the epithelium showed signs of persistent adhesion, both
clinically and histologically. In some animals, islets of ecto-
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PRELIMINARY EVALUATION OF THE BIOCOMPATIBILITY OF THE SMALL INTESTINAL SUBMUCOSA (SIS) 601

FIGURE 2. — Appearance of 7-mm diameter SIS grafts sutured into lamellar corneal beds of rabbit eyes :
(A) 2 days postoperatively ; (B) 4 days postoperatively ; (C) 10 days postoperatively ; (D) 15 days
postoperatively.

FIGURE 3. — Appearance of the cornea from rabbit 12, at postoperative

day 15. Neovascularization from corneoscleral limbus, mild residual
stromal oedema, and granulation tissue over the grafted area are
present.

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602 GRIGUER (F.) AND COLLABORATORS

FIGURE 4. — Histologic section of a rabbit cornea 7 days after SIS grafting. FIGURE 5. — Histologic section of a rabbit cornea 10 days after SIS grafting.
The entire graft is covered with new epithelium. Some epithelial cells are Stratification of the epithelial layers (Ep) has begun. There is prolifera-
entrapped within the graft (arrow). (H & E stain, 400 X). tion of keratocytes at the host-graft interface (arrowheads) and within the
graft (Gr) remnants. (H & E stain, 400 X).

FIGURE 6. — Histologic section of a rabbit cornea 15 days after SIS grafting. FIGURE 7. — Masson’s trichrome stained section of a rabbit cornea 15 days
A PAS postitive material yielding a distinct linear red staining (arrows) is after SIS grafting. The dark blue fibre bundles of the graft remnants
present along the epithelium-graft interface. Numerous keratocytes have (arrows) can be distinguished from the faint blue fibre bundles of the
colonized the graft remants (Periodic acid Schiff stain, 400 X). newly formed collagen (asteriks). (400 X).

FIGURE 8. — Histologic section of the cornea from rabbit 12. The SIS graft
is largely degraded and fragmented, and the newly formed collagen is
disorderly distributed. New vessels and an intense inflammatory infiltrate
of PMNs with occasional mononuclear cells have reached the grafted area
which is not completely covered with healed epithelium. (Masson’s
trichrome stain, 400 X).

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PRELIMINARY EVALUATION OF THE BIOCOMPATIBILITY OF THE SMALL INTESTINAL SUBMUCOSA (SIS)
pic epithelium were found within the stroma as a result of the
epithelial sliding inside and under the graft during the imme-
diate post-operative period. How the presence of ectopic epi-
thelium may affect the organisation of the stromal scar and
consequently the potential recovery of corneal integrity
remains to be determined. After 15 days, the SIS biomaterial
was partly substituted by a relatively clear corneal stroma,
and histologically the scar tissue was organised with kerato-
cytes and collagen fibres with an overall sagittal orientation,
parallel to the corneal surface. Since orientation of kerato-
cytes reflects the direction of repair and the potential reco-
very of corneal integrity [29], the regular arrangement of cor-
neal fibroblasts in the scar tissue suggests that lamellar conti-
nuity across the wound might be obtained over time and
result in a degree of stromal organisation close to normal. In
addition, an effect of SIS on the proliferative and migratory
behavior of stromal keratocytes is not unlikely since infiltra-
tion of the grafted material by numerous fibroblasts was
observed 10 to 15 days after surgery.
Several characteristics may explain why SIS biomaterial
allows for the reconstruction of the corneal epithelium and
stroma. Its epithelialisation is likely to be due to its collage-
nous nature, which ressembles the surface of a stromal wound
bed on which corneal epithelial tissue heals. Collagen I, one
of the components of the SIS extracellular matrix [8], is also a
biologic signal that was shown to allow rapid and sustained
epithelialisation in vivo [36]. Although this process has to be
further investigated, it is possible that SIS modifies the proli-
ferative and migratory behavior of stromal keratocytes and
thus the synthesis of collagen. Among the many factors that
can influence such processes, TGF-beta deserves special
attention because it can increase the synthesis of fibronectin
and collagens, and their accumulation around cells [14].
In conclusion, with limit of time of this experiment our
results suggest that SIS biomaterial causes minimal adverse
tissue reactions and allows corneal healing when used for
tectonic keratoplasty purpose. Therefore, we believe these
findings are encouraging and warrant further study to assess
the long term outcome of the grafted material with respect to
its potential use for corneal reconstruction.
Acknowledgments
The authors are most grateful to Françoise MICHAUD for
revising the manuscript and Pascal NIEZ for providing the
SIS biomaterial.
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