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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 RÉSUMÉ

The early stages of the ocular response to porcine small intestinal sub- Étude préliminaire de la biocompatibilité d’une greffe lamellaire de
mucosa (SIS) transplantation were assessed in a model of anterior keratec- sous-muqueuse intestinale dans la cornée du lapin. Par F. GRIGUER,
tomy in the rabbit. After surgery, the operated eyes (n = 17) were examined I. RAYMOND et A. REGNIER.
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 La réaction oculaire à une greffe cornéenne lamellaire de sous-muqueu-
biomaterial was well tolerated, and the corneal response was restricted to a se intestinale de porc (small intestinal submucosa : SIS) a été étudiée sur un
localized neovascularization. The grafted material was epithelialised within modèle de kératectomie superficielle chez le lapin. Après la chirurgie, un
7 days and incorporated into the corneal tissue without inducing granuloma suivi clinique des yeux opérés (n = 17) a été fait régulièrement pendant
formation. In one rabbit, the corneal inflammation was severe but the bio- 15 jours et les animaux ont été sacrifiés à des temps prédéterminés afin de
material was maintained in place. Although further studies with longer fol- réaliser une étude histopathologique des globes oculaires. Les résultats cli-
low-up periods are needed, these preliminary observations suggest that por- niques et histopathologiques montrent qu’à l’exception d’un cas, la greffe
cine SIS can be incorporated into the corneal tissue during healing process lamellaire de SIS a été bien tolérée et n’a entraîné qu’une néovascularisa-
and may represent a promising graft material for lamellar keratoplasty. 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.

KEY-WORDS : small intestinal submucosa - corneal tole- MOTS-CLÉS : sous-muqueuse intestinale - tolérance
rance - lamellar keratoplasty - rabbit. cornéenne - kératoplastie lamellaire - lapin.

Introduction primarily an acellular extracellular matrix composed of col-


lagen, proteoglycans, glycoproteins, and glycosaminogly-
The concept of a resorbable matrix replaced by autologous cans [8]. It also contains natural growth factors including
tissue is currently gaining acceptance as an effective proce- basic fibroblast growth factor (basic FGF) and transforming
dure for tissue reconstruction in numerous organ systems. growth factor beta (TGF-beta) [31].
This technique involves the use of a biodegradable scaffold
that the host tissue can use to remodel and regenerate. One SIS has several interesting physical and biological proper-
particular degradable biomaterial, small intestinal submu- ties : it has good tensile properties [33], is nonimmunogenic
cosa (SIS), has stimulated research investigation and clinical [5], is resistant to infection [6], and when used as a xenograft
interest in the last few years. SIS is derived from the submu- material promotes wound healing by providing a scaffold for
cosal layer of the porcine jejunum and consists of the stratum tissue ingrowth [24]. As a result, the collagen matrix is degra-
compactum layer of the tunica mucosa, the tunica muscularis ded and is replaced by an autologous tissue that closely
mucosa, and the tunica submucosa [5]. This biomaterial is resembles the native structure [5, 11].

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598 GRIGUER (F.) AND COLLABORATORS

Since 1989, xenogeneic porcine SIS has been successfully interrupted 10/0 nylon sutures. Chloramphenicol ophthalmic
used in laboratory animals for arterial or venous replacement ointment (Ophtalon®, TVM) was applied on the operated
[4, 6] and reconstruction of urinary bladder [21 22], cranial eye immediately following grafting and twice daily for
dura mater [12], meniscus [13], abdominal wall [11] or 7 days after surgery.
Achilles tendon [7]. Porcine SIS has recently become avai-
lable on the veterinary market and is currently used as a bio- CLINICAL FOLLOW UP
logical dressing for skin wounds in small animals and horses.
Although recent reports disclosed encouraging results with The recipient eyes were examined by slitlamp biomicro-
the use of porcine SIS for tectonic keratoplasty in one dog scopy on postoperative days 1, 2, 3, 4, 5, 6, 7, 10 and 15.
[26] and a small series of cats [15], the fundamental question According to their severity, conjunctival injection and che-
of SIS biocompatibility with the cornea remains unanswered. mosis, oedema of the peripheral cornea and iritis, characteri-
In an attempt to address this question, a study was designed zed by miosis and iridial hyperemia, were scored as follows : 0
to evaluate the early phase of the ocular response to a lamel- = absent ; 1 = mild ; 2 = moderate, and 3 = severe. The extent
lar grafting of SIS in a rabbit model of keratectomy wound. of corneal vascularization was assessed on the basis of the
Results from this study were reported as a clinical research farthest vascular ingrowth from the limbus as follows :
abstract at the British Small Animal Veterinary Association 0 = no corneal vascularization ; 1 = 2-mm vascular ingrowth ;
Congress in Birmingham, 6-9 April 2000. 2 = 4-mm vascular ingrowth, and 3 = 6-mm vascular
ingrowth. Biomicroscopic examination also evaluated the
integrity and appearance of the grafted material.
Materials and methods
HISTOPATHOLOGIC STUDIES
TEST MATERIAL
The animals were sacrified with an intravenous overdose
Under the lamellar flow hood, the grafts were prepared of barbiturate at 2 (n = 2), 4 (n = 2), 7 (n = 4), 10 (n = 3), and
from a sterile sheet of SIS (7.0 cm x 4.0 cm) available for 15 days (n = 6) postoperatively. After enucleation, the opera-
veterinary surgery (Vet Biosist®, Cook Veterinary Products). ted eyes were fixed in 10 % neutral buffered formaldehyde.
The dehydrated sheet was divided into discs, measuring The corneas with a rim of sclera were dissected from the fixa-
7.0 mm in diameter, with a corneal trephine. The discs were ted globes and mounted in paraffin. Sections 6 µm thick were
placed in sterile individual vials containing a sterile solution stained with haematoxylin and eosin (H & E), Masson’s tri-
for intraocular irrigation (Aqsia®, Chauvin Opsia SA) and chrome, and periodic acid Schiff (PAS) stains to be examina-
gentamicin at a concentration of 100 µg/ml (Gentalline® ted by light microscopy.
injectable 40 mg, Schering-Plough). The vials with the
enclosed disc of SIS were stored at 4°C until use.
Results
ANIMALS AND SURGICAL TECHNIQUE
Seventeen healthy male New Zealand White rabbits SURGICAL TECHNIQUE
without ocular disease and weighing 2.5 to 3 kg were used in In all cases, surgery was uneventful and technically not dif-
accordance with the ARVO Resolution on the Use of ficult because the SIS was tough and thick enough to hold the
Animals in Research. They were kept in individual cages 100 sutures without tearing.
under standard animal room conditions, fed dry pellets and
had water ad libitum. All rabbits were anesthetized intramus-
GROSS AND BIOMICROSCOPIC FINDINGS
cularly with 50 mg/kg body weight ketamine hydrochloride
(Kétamine 1000®, Virbac Animaux de Compagnie) and Postoperatively, none of the 17 rabbits showed evidence of
5 mg/kg body weight xylazine hydrochloride (Rompun®, discomfort and photophobia. Consistent degrees of conjunc-
Bayer Pharma). To supplement the general anesthesia, topi- tival injection and chemosis were evident in all operated eyes
cal 0.4 % oxybuprocaine (Novésine®, MSD-Chibret) was in the early postoperative period. The conjunctival injection
applied to the eye selected for grafting. and chemosis reached a maximum at day 1, with a mean
The eye to be operated was selected at random for each score of 1.65 and 1.35 respectively, and then gradually dimi-
rabbit. A pediatric eyelid speculum was used to expose the nished to disappear by days 10-15 (Fig. 1). In all but one ani-
globe and stay sutures of 4/0 silk were placed at 6 and 12 mal (rabbit 12), no corneal oedema was observed and the cor-
o’clock to stabilize the globe. Each animal underwent a cen- nea around the SIS graft remained transparent throughout the
tral lamellar keratectomy. A 7.0 mm corneal trephine with follow-up period. There was no evidence of stromal melting
depth adjustment to 0.2 mm was used to standardize the inci- around the keratectomy area in any of the operated eyes for
sion. The circumscribed epithelium and stroma were remo- the 15 days of the experimental period. In 11 of 13 (approxi-
ved with a Peaufique corneal dissector. A disc of SIS, similar mately 85 %) of the cases that were followed for at least
in size to the keratectomy, was removed from the storage 7 days, vessels could be seen invading the peripheral cornea
medium and placed over the corneal defect with its serosal from 4 to 6 days after surgery (Fig. 1). Vascular sprouts arose
side (tunica submucosa) facing the recipient bed. The graft from the circumlimbal vessels and formed a small band,
was secured to the edge of the corneal defect with 12 simple approximately 5 mm wide, in the superotemporal quadrant.

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PRELIMINARY EVALUATION OF THE BIOCOMPATIBILITY OF THE SMALL INTESTINAL SUBMUCOSA (SIS) 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.
FIGURE 1. — Effect of corneal implantation of SIS on individual parameters
of the ocular response in rabbits. Each point represents mean score for In all but one (rabbit 12) specimens obtained on the 10th
parameter on designated day of postoperative follow-up (see text for sco- and 15th postoperative days, a mild residual inflammatory
ring procedures). Vertical lines indicate 1 SD.
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
The neovessels were superficial and few in number. They layer of basal cells that were cuboidal and lightly stained
progressively grew, perpendicular to the limbus and toward (Fig. 5). Activated keratocytes were present in great number
the region of the graft, to a length of 2-4 mm which persisted just under the grafted material and were identified between
throughout the experiment. Moderate signs of iritis, manifes- the collagen fibres of the graft remnants (Fig. 5). By 15 days
ted as iris vascular injection and/or miosis, were present in all post-grafting, PAS staining sections showed that a new base-
eyes for the first 4-6 postoperative days (maximum mean ment membrane was present under the epithelium (Fig. 6). At
score = 1.53) and subsequently regressed without sequela that time, the corneal thickness was restored and the newly
(Fig. 1). formed collagen fibres appeared more regularly oriented than
During the first 48 hours following surgery, there was no those of the graft remnants and grossly parallel to the corneal
visible change in the appearance of the grafts (Fig. 2A). From surface (Fig. 7). Histology of the cornea from rabbit 12 sho-
postoperative days 3-4 to 6-10, the xenografts appeared opa- wed a severe infiltration of the grafted material and surroun-
lescent and swollen (Fig. 2B). Their superficial layers were ding stroma by PMNs and a few mononuclear cells.
deliquescent and sloughed off, forming small whitish debris Remnants of the SIS appeared fragmented and disorganised,
at the surface of the grafted area (Fig. 2C). Nevertheless, all and epithelial coverage of the graft was incomplete (Fig. 8).
grafts were retained on the wound bed and became progressi- At each time point of the follow-up, the other structures of
vely less opaque by postoperative days 8-10. In parallel, their the anterior and posterior segments appeared to be histologi-
surface became smoother and brighter. By postoperative day cally normal.

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600 GRIGUER (F.) AND COLLABORATORS

Discussion tive barrier against the tear film and consequently did not pre-
vent the influx of PMNs from tears. In the other one, soluble
Deep ulcers are a common problem in small animal oph- components within SIS might act as chemoattractants to sti-
thalmology and require prompt diagnosis and adequate treat- mulate the inflammatory reaction. As such, TGF-beta is
ment. In addition to medical therapy a number of surgical known to have chemotactic properties to stimulate corneal
options can be used, each having both advantages and limita- leukocytic infiltration [32] and angiogenesis [27]. In the cur-
tions. Conjunctival grafts are probably the most commonly rent study, the leukocyte infiltration preceded neovasculari-
used surgical techniques. Although these procedures have zation and initiation of angiogenesis was very likely a conse-
been shown to be effective, they can also lead to scarring and quence of the leukocytic infiltration, since corneal angioge-
pigmentation of the recipient cornea [18]. Corneoscleral nesis is reportedly facilitated by the presence of leukocytes in
transposition, autologous sliding lamellar keratoplasty, or the stroma [28]. This response might be amplified by a
homologous lamellar corneal transplantation is also useful variety of growth factors contained in SIS. For instance, it
when there is a risk of perforation, as each of these tech- has been shown that basic FGF can stimulate corneal angio-
niques is aimed to replace the damaged stroma and promote genesis without inducing an inflammatory reaction [19].
healing with re-epithelialisation [16, 35]. These three tech- The ocular reaction following SIS grafting was severe in
niques have the advantages to allow for better cosmetic only one operated eye (rabbit 12) and overall, many of the
appearance and functional vision than conjunctival grafts clinical features of this case paralleled those found in corneal
[16]. As the use of fresh or preserved homologous lamellar xenograft rejection [9]. Nevertheless, the SIS graft was main-
grafts may be limited by the difficulty of harvesting and/or tained in place, and the ocular inflammation became less
storing donor tissue preparation [17], some investigators intense 10 days after surgery. Histologically, the components
have evaluated the possibility of repairing corneal ulcers with of the inflammatory infiltrate consisted primarily of PMN
various biological membranes that are more readily available leucocytes with fewer lymphocytes and plasma cells. These
than homologous corneal grafts. For instance, preserved histopathological features are different from those observed
homologous amniotic membrane has been used in humans to in rejecting corneas, that are characterized by an accumula-
treat deep corneal ulcers [25], and transplantation of preser- tion of lymphocytes and monocytes [30]. Although current
ved xenologous pericardium [2], renal capsule [1] or amnio- speculation is that there is no inherent danger of an immuno-
tic membrane [3] was a successful treatment of experimental logic reaction, because SIS biomaterial does not contain live
corneal defects in dogs. Despite these results, major limita- cells [23], our observation raises the question whether SIS
tions for clinical application of these techniques appear to be may elaborate an immunogenic response when implanted in
the preparative and storage procedures to which the trans- the cornea. Further studies will be needed to elucidate this
plants should be submitted before use [25]. Such drawbacks hypothesis. Pre-operative SIS biomaterial contamination or
might be eliminated by using a biomaterial ready to use, such post-operative infection may be other possibilities to explain
as the SIS marketed for veterinary surgical applications. the inflammatory reaction in this animal. Although these
In the present study, the lamellar grafting of SIS was found hypotheses cannot be dismissed, the risk of bacterial conta-
to produce a mild-to-moderate degree of inflammatory mination was minimized by adding gentamicin to the storage
adverse reactions, both clinical and histopathologic. solution and applying chloramphenicol ophthalmic ointment
Transient conjunctival inflammation and iritis were consis- during the first postoperative week.
tently observed in the early postoperative phase. This non This study also examined the early phases of corneal epi-
specific response was likely due to the trauma of surgery thelium and stromal reconstruction following SIS transplan-
since it is well known that the rabbit eye has a high sensitivity tation, and our preliminary results suggest that this biomate-
to the surgical trauma and rapidly responds by an inflamma- rial was incorporated into the cornea during a wound-healing
tory reaction, including conjunctival hyperemia, chemosis, response. During the first postoperative days, the SIS graft
iridial hyperemia, and miosis [10, 34]. With the exception of seems to serve both as a patch and a graft for re-establish-
one case, no obvious symptoms of graft rejection were obser- ment of the epithelial cells because epithelialisation can take
ved since the corneas remained clear throughout the follow- place over, inside and underneath the grafted material.
up period and had a developing reaction to SIS that was Although the biomaterial was secured to the keratectomy
confined to a moderate neovascularization. Neovessels were wounds with the more densely compacted side (stratum com-
produced from a small area of the peripheral cornea and were pactum) external, epithelialisation of the graft from neighbo-
apparent in 3 to 6 days. This rate of vascularization coincides ring corneal epithelium was not facilitated in the first post-
with other reported observations on corneal angiogenesis operative days since the newly formed epithelium was elimi-
induced by xenografts in rats [9] and biodegradable polymers nated with the superficial layers of the graft. In comparison,
in rabbits [19]. Histologically, a slight degree of PMN infil- a 7 mm keratectomy wound in the rabbit is spontaneously
tration was observed around the keratectomy area in the early covered by epithelial cells in approximately 66 hours [37]. In
postoperative days. There was no evidence of granuloma for- the current study, the epithelium was reestablished above the
mation, a finding which is coherent with previous reports on grafted material at day 7 and stratified from days 10 to 15. At
body wall [11] and bladder wall response [20, 22] to SIS this early stage after grafting, the number of cell layers was
implantation. It is likely that there were at least two routes for less than in a normal rabbit corneal epithelium but despite
the leukocytic infiltration. In the first one, the grafted mate- this, the epithelium showed signs of persistent adhesion, both
rial, although sutured to the cornea, did not provide an effec- 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) 603

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