Canadian Patents Database / Patent 2055099 Summary

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(12) Patent Application: (11) CA 2055099
(54) English Title: AMNIOTIC MEMBRANE GRAFT OR WRAP TO PREVENT ADHESIONS OR BLEEDING OF INTERNAL ORGANS
(54) French Title: GREFFON OU ENVELOPPE DE MEMBRANE AMNIOTIQUE POUR PREVENIR LES ADHERENCES OU LE SAIGNEMENT DES ORGANES INTERNES
(51) International Patent Classification (IPC):
  • A61L 27/00 (2006.01)
  • A61F 2/10 (2006.01)
(72) Inventors :
  • YOUNG, RONALD L. (United States of America)
(73) Owners :
  • RESEARCH DEVELOPMENT FOUNDATION (United States of America)
(71) Applicants :
(74) Agent: BORDEN LADNER GERVAIS LLP
(74) Associate agent: BORDEN LADNER GERVAIS LLP
(45) Issued:
(22) Filed Date: 1991-11-07
(41) Open to Public Inspection: 1993-05-08
(30) Availability of licence: N/A
(30) Language of filing: English

English Abstract



Amniotic Membrane Graft or Wrap to Prevent
Adhesions or Bleeding of Internal Organs
Abstract of the Disclosure
Disclosed is a specially treated amniotic membrane
which prevents adhesions following injury, such as surgical
injury and prevents bleeding of internal organs caused by
injury. The amniotic membrane has its cellular monolayer on
its fetal side removed by treatment with trypsin and is
sterilized by gamma irradiation which also cross-links its
collagen. It is applied as a single layer to an injured
surface subject to adhesions or wrapped on internal organs
subject to bleeding, and can be sutured or applied by a
liquid anti-adhesive adjuvant. The graft can be applied to
a wet as well as a dry surface.


Note: Claims are shown in the official language in which they were submitted.


-12-
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A graft for preventing adhesions following injury
in a patient comprising,
a sheet of amniotic membrane for application on a
surface of the patient subject to the formation of the
adhesions,
the amniotic membrane having its cellular monolayer
on its fetal side removed by treatment with trypsin,
and
sterilized and having its collagen cross-linked by
gamma irradiation.

2. The graft of Claim 1 where,
the radiation is a minimum of 0.20 M rads.

3. The graft of Claim 1 where,
the range of irradiation is from 0.25 M to 2.5 rads.

4. The graft of Claim 1 where,
the treatment with trypsin is a 10% solution by
volume.

5. The graft of Claim 1 where,
the sheet of amniotic membrane is a single layer.

6. The graft of Claim 2 where,
the sheet of amniotic membrane is a single layer.

7. The graft of Claim 3 where,
the sheet of amniotic membrane is a single layer.

8. The graft of Claim 4 where,
the sheet of amniotic membrane is a single layer.

9. A method of preparing a graft for preventing



-13-
adhesions in a patient due to injury comprising,
removing cellular material on its fetal side from an
amniotic membrane from human placenta by treatment with
trypsin, and
sterilizing and cross-linking collagen of the
membrane by gamma irradiation.

10. The method of Claim 9 where,
the irradiation is a minimum of 0.20 M rads.

11. The method of Claim 9 where,
the irradiation is in the range of 0.25 M to 2.5 M
rads.

12. The method of Claim 9 where,
the amniotic membrane is a single layer.

13. A method of preventing adhesions following injury
in a patient comprising,
applying the graft of Claim 1 on the surface subject
to the formation of the adhesions.

14. A method of preventing adhesions following injury
in a patient comprising,
applying the graft of Claim 2 on the surface subject
to the formation of the adhesions.

15. A method of preventing adhesions following injury
in a patient comprising,
applying the graft of Claim 3 on the surface subject
to the formation of the adhesions.

16. A method of preventing adhesions following injury
in a patient comprising,
applying the graft of Claim 4 on the surface subject
to the formation of the adhesions.


-14-
17. The method of Claim 14 where,
the method of applying the graft is by suturing.

18. The method of Claim 14 where,
the method of applying the graft is by a liquid
anti-adhesive adjuvant.

19. The method of preventing bleeding of an internal
organ comprising covering at least the bleeding portion of
the internal organ with the graft of Claims 1, 2, 3, 4, 5,
6, 7, or 8.

Note: Descriptions are shown in the official language in which they were submitted.

2C`~ 9




Amniotic Membrane Graft or Wrap to Prevent
Adhesions or Bleeding of Internal Oraans

Field of the Invention
The present invention is in the field of preventing
adhesions following injury, and particularly following
surgical injury, and to preventing bleeding of internal
organs following injury.
Backaround of the Invention
Adhesions may result from a great number of medical
conditione or from surgical intervention. Illness leading
to adhesion formation includes pelvic inflammatory or other
pelvic or abdominal inflammatory processes resulting either
from infection or endometriosis. The surgical procedures
required by these and other pathologic conditions, e.g.
cysts, tumors, etc., may also result in adhesion formation.
Adhesions may, in turn, be associated with infertility by
causing occlusion of the fallopian tubes or by interfering
with tubal/ovarian function, inhibition of ovum pickup being
the best example. It is postulated that the formation of
adhesions evolves from trauma to serosal surfaces followed
by release of a fibrinogen-rich exudate and preceding on to
fibrin formation. This leads either to thick or filmy
adhesive bands which may bridge the pelvic organs or tissues
or to the dense fixation of these structures to each other.
The presence of pelvic or abdominal adhesions is known to be
a major cause of infertility in the human female.
Through the years a great number of natural and
synthetic graft materials has been employed in an effort to

2~ 39


reduce adhesion formation on traumatized surfaces, however,
with only marginally successful results. Natural materials
have included peritoneum, omentum, fat, and amnion, as well
as amnion plus chorion. Synthetic materials including
polyvinyl alcohol film and tantalum foil were used in the
past, and, more recently, barriers consisting of Gelfilm and
Gelfoam paste (Upjohn Co., Kalamazoo, Michigan), Surgicel
(Johnson & Johnson, New Brunswick, New Jersey), and Silastic
(Dow-Corning, Midland, Michigan), as well as meshes of Gore-
Tex (Gore-Tex, Gore, Texas) and Interceed (Johnson &
Johnson, New Brunswick, New Jersey) have been employed. The
newer materials have led to more promising results.
Injury to internal organs, such as the liver, pancreas,
spleen, kidney, and the like resulting in bleeding has been
a problem over the years resulting in many cases in loss of
the organ and death because of ineffective means and methods
of preventing such bleeding.
The use of human amnion as a surgical adjunct has a
long history. An excellent review has been published by
Trelford and Trelford-Sauder. Amnion has been tried
unsuccessfully to prevent pelvic and abdominal adhesions in
a number of experimental animal models, as well as in human
patients. The natural membrane has been tried in tubal
surgery and there exists an extensive experience with its
use in vaginal reconstructive surgery in women. Other
applications include repair of conjunctival defects,
reconstruction of the bile duct, and prevention of
meningocerebral adhesions following craniotomy. Its primary
role in humans, however, has remained in the areas of burns,
ulcers and other skin trauma and in wound healing.
Prior to the present invention, no substantial
literature existed describing the potential of human amnion
in preventing intra-abdominal adhesions in humans, although
some progress has been made in this area through the use of
a number of synthetic agents. ~3adaway, et al. recently
reported on the intra-abdominal application of amniotic

Z(~5~39

membranes to prevent adhesions in the rat model. They noted
little effect in inhibiting adhesion formation on serosal
surfaces but observed somewhat better results on the
parietal peritoneum. The explanations which they offered
for the lack of success involved problems with post-
operative organ immobility and blood pooling, both of which
may play a role in adhesion formation after human surgery.
No substantial literature exists describing wrapping of
internal organs with amniotic membranes to prevent bleeding
following injury to injured internal organs such as the
liver, pancreas, spleen, kidney and the like.
A study of the literature reveals almost as many
different methods of preparing and storing the membranes as
there are case or experimental reports. Poor results were
obtained with glutaraldehyde-treated membranes (unpublished
data), as well as equally unsatisfactory experience
elsewhere with alcohol pretreatment and oven drying or
simple freezing in saline. Unexpectedly, pretreatment of
the amni~tic membrane by trypsin washing and gamma
irradiation according to the present invention is successful
in preventing adhesions. The membranes thus prepared
underwent adequate neovascularization and caused no
significant inflammatory infiltration. This also supports a
conclu~ion of no significant immunological reaction induced
by the membranes, as also observed in the Badaway study.
It would be highly advantageous to provide amniotic
membranes from human placenta which successfully prevent
adhesions following injury, such as following surgical
ln~ury .
It would be highly advantageous to provide wrapping or
covering of injured bleeding organs with amniotic membranes
which successfully stops the bleeding.
Summary of the Invention
The present invention is directed to specially prepared
amnion membranes, their process of making and use which
successfully prevent adhesions due to injury, such as

~5~'~9
--4--
surgical injury or injuries to internal organs which
successfully prevents bleeding.
The amniotic membranes are obtained from fresh human
placentas. The amnion layer is separated from the placenta.
Cellular monolayer material overlying the basal lamina on
the fetal side of the membrane is removed by soaking in a
solution of trypsin. The amnion is rinsed repeatedly with
phosphate buffer solution or distilled water until cleaned
and then exposed to 2.5 M rads of gamma irradiation which
cross-links the collagen, sterilizes the tissue, provides
protection against viral disease transmission, strengthens
and permits use of the amnion in single layer form.
Preferably, the entire membrane is irradiated at 60,000 rads
per hour for 20 minutes. The membranes are then cut into
smaller squares of approximately 2 x 2 cm and re-radiated at
60,000 rads per hour for a total of 33 hours 20 minutes
equivalent to a 2,000,000 rad dose. The small squares thus
prepared were frozen at -70C in distilled water to maintain
them until they were used (within 4 weeks). Just prior to
use, the membranes were thawed at 22C. No antibodies were
used during this process.
The thinness and the exceptional compliant quality of
the membranes made them extremely facile to use. Single
layer application of the membrane is mandatory in the pelvis
and abdomen to prevent fibrosis formation within the
membrane itself. Although the synthetic grafts are applied
without suturing, these membranes can be fixed in place
using microsutures or the use of liquid anti-adhesive
adjuvants such as dextran. An added technical advantage to
the use of these amniotic membranes as opposed to the
synthetic meshes is the fact that they can be applied and
sutured over surfaces not perfectly dry.
Accordingly, it is an object of the present invention
to provide a graft or wrap of trypsin-treated, gamma
irradiated amniotic membrane from human placenta for
preventing adhesions in a patient following injury or

2C`~:5~


bleeding of internal organs.
It is a further object of the present invention to
provide such a graft or wrap in which the cellular monolayer
on the fetal side of the amniotic membrane is removed by
trypsin.
It is a further object of the present invention to
provide such a graft or wrap in which the amniotic membrane
having its cellular monolayer material removed is sterilized
and has its collagen cross-linked by gamma irradiation.
lo It is a further object of the present invention to
provide a method of preparing a graft or wrap for preventing
adhesions or bleeding of internal organs in a patient due to
injury in which cellular material on the fetal side of the
amniotic membrane is removed by treatment with trypsin, and
the membrane is sterilized and its collagen cross-linked by
gamma irradiation.
It is still a further object of the present invention
to provide a method of preventing adhesions or bleeding of
internal organs following injury in a patient, comprising
applying a trypsin-treated, gamma irradiated amniotic
membrane to the surface of a patient subject to the
formation of adhesion or bleeding.
other and further objects, features and advantages
appear throughout the specification and claims.
Description of Preferred Embodiments
The graft of the invention comprises a trypsin-treated,
gamma irradiated, human single layer amniotic membrane which
successfully prevents adhesions and preventing bleeding
following injury, such as surgical injury. Excellent
results have been obtained by employing these treated single
layered grafts.
The method of preparing these amniotic membrane grafts
according to the invention comprises harvesting freshly
delivered human placentas, preferably taken at the time of
cesarean section, manually separating the amniotic membranes
from the chorion, washing in distilled water, treating the

2~ 9

cleaned amniotic membranes treated by soaking in a solution
oE trypsin to remove cellular material from the fetal side,
and then sterilizing and cross-linking the collagen of the
amniotic membrane by gamma irradiation.
The method of preventing adhesions and bleeding of
internal organs according to the invention comprises
applying a single layer sheet of the graft to the injured
surface with or without suturing or by use of liquid anti-
adhesive adjuvants such as dextran. Advantageously, these
amniotic membrane grafts and wraps can be applied and
sutured over surfaces not perfectly dry.
EXAMPLE 1
Materials and Methods
The amniotic membranes were harvested from freshly
delivered human placentas taken at the time of cesarean
section. The amniotic membranes were manually separated
from the chorion and washed in distilled water. The clean
membranes were first treated by soaking for three hours in a
10% solution of trypsin. Subsequently, they were irradiated
with gamma irradiation to sterilize them in the following
mannerO First, the entire membrane underwent an 8 hour 20
minute irradiation at 60,000 rads per hour for a total dose
of 500,000 rads. The membranes were then cut into smaller
squares of approximately 2 x 2 cm and reradiated at 60,000
rads per hour for a total of 33 hours 20 minutes equivalent
to a 2 million rad dose. The small squares thus prepared
were frozen at -70C in distilled water to maintain them
until they were used (within 4 weeks). Just prior to use,
the membranes were thawed at 22C. No antibiotics were used
during this process.
Surgical Procedure
Study animals consisted of multiparous female New
Zealand rabbits each weighing at least 3.5 kilograms to
ensure adequate size of the pelvic organs. Six rabbits were
assigned to each group A through C, and 18 to group D. At
the time of surgery the rabbits were anesthetized with a

z~

mixture consisting of ketamine, promazine, and xylozine at a
dosage of 1 cc/kg of body weight. The abdomen was shaved,
subjected to sterile preparation and draped. Sterile
microsurgical techniques under the operating microscope were
employed as previously described by Badaway, et al.
Experimental injuries consisted of a series of incisions
through the serosal and muscularis layers of the uterine
horn extending into the endometrial cavity with frequent
avulsion of the mucosa. The cuts, 1 cm long and spaced 5
millimeters apart, were created with microscissors.
Membrane grafts approximately 1 x 2 cm in size were sutured
into place over the lesion in a single layer using multiple
interrupted sutures of 7-0 maxon. The maternal side of the
membrane was placed against the injury, and the fetal side
faced into the abdominal cavity. Following surgery, the
abdomen was closed in three layers and a sterile dressing
left in place for 72 hours. All animals received procaine
penicillin at a dosage of 50 ml per kg IM qOD x 5 doses
postoperatively and were maintained in a vivarium at 27C
with 40-70% humidity and given Purina Rabbit Chow pellets
and water ad libitum.
Description of Experimental Groups
Animals were randomly assigned to 3 groups of 6 animals
each and a fourth group of 18 animals. Each rabbit was
subjected to 2 surgical procedures, the initial laparotomy
including the designated operative procedure, and a second
look laparotomy to evaluate the effects of the experimental
intervention. The groups were as follows: Group A (n + 6)
was the background control group. The abdomen in this group
was opened, exposed to no specific injury or treatment and
closed. Group B (n = 6) was the model control group in
which controlled injuries, as described above, were made on
one uterine horn of each animal. The contralateral horn was
not injured, and no therapeutic interventions were made on
either horn. Group C (n = 6) was the first treatment group
where injuries were carried out on one uterine horn as in

2~5~

Group B, and then both injured and non-injured horns were
covered with membrane grafts held in place with
microsutures. Group D tn=16) formed the second treatment
group in which both uterine horns were experimentally
injured in a similar manner. One horn was then treated by
suturing a membrane into place, and the contralateral horn
was treated with interrupted, hemostatic microsutures of 7-0
maxon.
Thirty days after the initial laparotomy and surgical
intervention each animal was reoperated, and adhesions were
photographed and evaluated with regard to their presence or
absence, percentage of surface included, and graded as to
adhesion quality (thin, thick, filmy, dense). Statistical
analysis was performed using Fisher's Exact Test. It was
considered significant at p < 0.05.
Results
All results are summarized in Table 1.

2~ 9

_g


Table 1 Comparison of Rcsults in Membrane-Treated
Versus Untreated Uterine Hours Following Experimental Injury

Group Horn Surgical Number with Types of Percentage ot
Procedure Adhesions (%) Ahesions Sur~ace Affected

A R none û
n=ô L none O

0 B R Inclsions 6 "'l dense, thick 75
-a
n=ô L none O

C R Incisions ~ O
1 5 membrane
n=B L Membrane 1 (17) tilmy,thin 10
only

D R Inclslon8 + 3 ~17) r fllmy, thln 10
2 0 mombrane
a
n-18 L Inslclons + 18 (100) den8e,thick 80
sutures

2 5 a Rl~ht vs left horn, Flsher'8 Exact Test. In each case p < 0.05.

The background control group plus model control Group B
confirmed the validity of the model by demonstrating that
there were no backqround adhesions from laparotomy alone and
that the experimental injury was sufficient to cause dense
adhesions in 100% of the cases if untreated. These included
surface adhesions as well as loop-to-loop adhesions leading
to severe tortuosity of the involved horn. It was further
noted that there was no crossover of these adhesions to the
injured contralateral horn (p = 0.002).
Experimental Group C showed no significant difference
(p = 0.5) in adhesion formation on sites of membrane grafts

--10--
placed over injured versus non-injured uterine horns. Thin,
filmy adhesions were found in only one case on the non-
injured horn and in no cases on the injured side. Finally,
in experimental Group D, membrane grafts significantly
reduced the formation of adhesions as compared to those
found at the site of hemostatic microsutures. Dense, thick
adhesions over an average of 80% of the surface area of the
sites of injury were noted on the horns treated with the
sutures in 100% of the cases. In contrast, only 17~ of the
injured horns (p = 0.0000003) which had been covered with
membrane grafts showed any adhesions, and these were thin,
filmy, and covered only about 10% of the injured/grafted
area. Histological studies showed that the membranes were
integrated with the serosal layer and showed
neovascularization at the site of the graft. Minimal
polymorphonuclear infiltration of the serosal surfaces was
present suggesting no significant immunological response.
The thinness and the exceptional compliant quality of
the single layer membranes made them extremely facile to
use. Single layer application is mandatory in the pelvis
and abdomen to prevent fibrosis formation within the
membrane itself. Also, the synthetic grafts are applied
without suturing, and in the foregoing examples the
membranes were fixed in place using microsutures. In the
human, this adds the potential for concomitant use of liquid
anti-adhesive adjuvants such as dextran. Also, an added
technical advantage to the use of these amniotic membranes,
as opposed apparently to the synthetic meshes is the fact
that they can be applied and sutured over surfaces not
perfectly dry.
EXAMPLE 2
In this example, formation of adhesions involving the
parietal peritoneum was prevented by these membranes as
prepared and as used in Example 1 which improves the outcome
of procedures involving extensive endometriosis or pelvic
sidewall adhesions of the adnexal structures.

2C`5~39


EXAMPLE 3
In this example, sheets of the amniotic membranes are
wrapped about or cover injured and bleeding portions of
internal organs, the liver, pancreas, spleen, kidney and
successfully prevent such bleeding. The amniotic membranes
are applied into place as in the preceding examples. As
previously mentioned, these amniotic membrane grafts and
wraps can be applied and cover wet surfaces, as well as dry
ones.
The foregoing animal studies are excellent models for
prevention of adhesions and bleeding of internal organs in
human beings. The term "patient" as used herein includes
human and animal patients.
The present invention, therefore, is well suited and
adapted to attain the objects and ends and has the features
and advantages mentioned as well as others inherent therein.
While presently preferred embodiments have been given
for the purpose of disclosure, changes can be made therein
which are within the spirit of the invention as defined by
the scope of the appended claims.

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Admin Status

Title Date
Forecasted Issue Date Unavailable
(22) Filed 1991-11-07
(41) Open to Public Inspection 1993-05-08
Dead Application 1997-11-07

Abandonment History

Abandonment Date Reason Reinstatement Date
1996-11-07 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Filing $0.00 1991-11-07
Registration of Documents $0.00 1992-06-02
Maintenance Fee - Application - New Act 2 1993-11-08 $100.00 1993-10-15
Maintenance Fee - Application - New Act 3 1994-11-07 $100.00 1994-10-18
Maintenance Fee - Application - New Act 4 1995-11-07 $100.00 1995-10-12
Current owners on record shown in alphabetical order.
Current Owners on Record
RESEARCH DEVELOPMENT FOUNDATION
Past owners on record shown in alphabetical order.
Past Owners on Record
YOUNG, RONALD L.
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.

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Drawings 1993-05-08 1 6
Claims 1993-05-08 3 72
Abstract 1993-05-08 1 20
Cover Page 1993-05-08 1 14
Description 1993-05-08 11 487
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Fees 1994-10-18 1 74
Fees 1993-10-15 1 61