CA2055099A1 - Amniotic membrane graft or wrap to prevent adhesions or bleeding of internal organs - Google Patents

Amniotic membrane graft or wrap to prevent adhesions or bleeding of internal organs

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Publication number
CA2055099A1
CA2055099A1 CA 2055099 CA2055099A CA2055099A1 CA 2055099 A1 CA2055099 A1 CA 2055099A1 CA 2055099 CA2055099 CA 2055099 CA 2055099 A CA2055099 A CA 2055099A CA 2055099 A1 CA2055099 A1 CA 2055099A1
Authority
CA
Canada
Prior art keywords
graft
adhesions
method
amniotic membrane
bleeding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA 2055099
Other languages
French (fr)
Inventor
Ronald L. Young
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Research Development Foundation
Original Assignee
Research Development Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Research Development Foundation filed Critical Research Development Foundation
Priority to CA 2055099 priority Critical patent/CA2055099A1/en
Publication of CA2055099A1 publication Critical patent/CA2055099A1/en
Application status is Abandoned legal-status Critical

Links

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.

Description

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 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.

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 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.

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 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.

Claims (19)

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 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.
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.
CA 2055099 1991-11-07 1991-11-07 Amniotic membrane graft or wrap to prevent adhesions or bleeding of internal organs Abandoned CA2055099A1 (en)

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Application Number Priority Date Filing Date Title
CA 2055099 CA2055099A1 (en) 1991-11-07 1991-11-07 Amniotic membrane graft or wrap to prevent adhesions or bleeding of internal organs

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Application Number Priority Date Filing Date Title
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0637452A1 (en) * 1993-08-06 1995-02-08 Yasuhiko Shimizu Collagen membrane material for medical use and process for preparing the same
EP0669138A2 (en) * 1994-02-24 1995-08-30 Research Development Foundation Amniotic membrane graft or wrap to prevent adhesions or bleeding of internal organs
EP0734736A1 (en) * 1995-03-31 1996-10-02 Toyo Boseki Kabushiki Kaisha Medical device and method for producing the same
US5607590A (en) * 1993-08-06 1997-03-04 Shimizu; Yasuhiko Material for medical use and process for preparing same
EP0773033A1 (en) * 1995-10-31 1997-05-14 Bio-Engineering Laboratories, Ltd. A raw membranous material for medical materials and manufacturing methods thereof
EP0773032A1 (en) * 1995-10-31 1997-05-14 Bio-Engineering Laboratories, Ltd. Medical materials and manufacturing methods thereof
WO2003020327A2 (en) * 2001-09-06 2003-03-13 Bone Sa A cross-linked collagenous biomaterial

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0637452A1 (en) * 1993-08-06 1995-02-08 Yasuhiko Shimizu Collagen membrane material for medical use and process for preparing the same
US5607590A (en) * 1993-08-06 1997-03-04 Shimizu; Yasuhiko Material for medical use and process for preparing same
EP0669138A2 (en) * 1994-02-24 1995-08-30 Research Development Foundation Amniotic membrane graft or wrap to prevent adhesions or bleeding of internal organs
EP0669138A3 (en) * 1994-02-24 1996-02-14 Res Dev Foundation Amniotic membrane graft or wrap to prevent adhesions or bleeding of internal organs.
EP0734736A1 (en) * 1995-03-31 1996-10-02 Toyo Boseki Kabushiki Kaisha Medical device and method for producing the same
US5723010A (en) * 1995-03-31 1998-03-03 Toyo Boseki Kabushiki Kaisha Medical device and method for producing the same
EP0773033A1 (en) * 1995-10-31 1997-05-14 Bio-Engineering Laboratories, Ltd. A raw membranous material for medical materials and manufacturing methods thereof
EP0773032A1 (en) * 1995-10-31 1997-05-14 Bio-Engineering Laboratories, Ltd. Medical materials and manufacturing methods thereof
US5916266A (en) * 1995-10-31 1999-06-29 Bio-Engineering Laboratories, Ltd. Raw membranous material for medical materials and manufacturing methods thereof
WO2003020327A2 (en) * 2001-09-06 2003-03-13 Bone Sa A cross-linked collagenous biomaterial
WO2003020327A3 (en) * 2001-09-06 2004-06-10 Bone Sa A cross-linked collagenous biomaterial

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