JPH10113384A - Medical substitute membrane and manufacture therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medical substitute membrane which can prevent adhesion of an operative wound by arranging a gelatin gel layer or a hyaluronic acid layer on at least one outside surface of a laminated body in the laminated body having a porous intermediate material composed of a sheet-like biodegradable absorptive material between collagen membranes of two layers. SOLUTION: A porous intermediate material composed of a sheet-like biodegradable absorptive material, is sandwiched by collagen membranes of two layers through an adhesive, and is formed as a laminated body, and after this laminated body is submitted to first cross-linking processing, a gelatin gel layer of a hyaluronic acid layer is formed on at least one outside surface of the laminated body, and afterward, the laminated body is submitted to second cross-linking processing, and a medical substitute membrane is obtained. In this case, a membrane composed of a natural collagen membrane derived from a human being, neutral soluble collagen, acid soluble collagen, alkali soluble collagen or the like, is used as the collagen membranes. A material selected form a copolymer or the like with a polyglycoilic acid, a polylactic acid, a glycolic acid and a lactic acid is cited as the porous intermediate material.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a medical substitute membrane, and more particularly, to prevent adhesion between biological parts by filling a defective part of a biological membrane such as brain dura, pericardium, pleura, peritoneum or serosa. The present invention relates to a medical substitute membrane that can be used and a method for manufacturing the same.

[0002]

2. Description of the Related Art Due to various diseases or trauma, etc., surgical operation of the brain and various organs is performed, and when the surgical wound is closed, the incised cerebral dura, pericardium, pleura, peritoneum or serosa is resewn. It is necessary to close it, but the shortening due to the sewing margin occurs,
Since the membrane is partially resected, it cannot be completely closed and often has a defect in the membrane. If such a defect is left as it is, the brain, heart, lungs,
Since the intestine and other organs adhere to surrounding tissues, the tissues are damaged and a good prognosis cannot be obtained. For this reason, conventionally, as a material for filling this defective portion, for the brain dura, freeze-dried human brain dura collected from the cadaver,
A porous stretched polytetrafluoroethylene film material (EPTFE) (Goretex for tissue, registered trademark) is used, and a copolymer of lactic acid and ε-caprolactone (50:50) is currently being developed. . For the pericardium, EPTFE material, bovine pericardium, equine pericardium and the like are also used. At present, no pleural or peritoneal membrane is used as an alternative membrane.

[0003] However, the use of human dura is not only disadvantageous in that the supplemented material and the parenchyma tissue adhere to each other, and may cause a seizure of tencan after the operation. There are ethical issues with harvesting, very limited supply, and more recently, Creutzfeldt-Jakob Disease ( (CJD) has been reported (Neurosurgery, 21 (2): 167-170, 1).
993). Further, since the EPTFE material is not decomposed in a living body and remains as a foreign substance, when it comes into contact with a living tissue, the tissue cells undergo fat degeneration. A copolymer of lactic acid and ε-caprolactone is biodegradable, and gradually decomposes after application to a living body, but it takes a long time of about one year until it is decomposed and absorbed. As such, it may also remain in the body for some time as a foreign body, causing inflammation of the tissue and forming granuloma. This copolymer is
Since (L) -form lactic acid is blended, lactic acid may crystallize in the copolymer and cause inflammation. Also, EP
Neither TFE nor the copolymer of lactic acid and ε-caprolactone has an effect of promoting regeneration of a biological membrane.

[0004]

Therefore, it is supplied stably without any ethical problems, and after application to a living body, prevents adhesion of surgical wounds after surgery, has no fear of infection, and has no risk of infection. Therefore, there has been a demand for the development of a material which can control the rate of decomposition after application without causing the denaturation of the protein and which desirably promotes the regeneration of a biological membrane, particularly the dura mater, pericardium, pleura, peritoneum or serosa.

[0005]

SUMMARY OF THE INVENTION The present invention is a laminate comprising a sheet-like porous intermediate material made of a biodegradable and absorbable material via an adhesive between two layers of collagen membrane, The present invention relates to a medical substitute film having a gelatin gel layer or a hyaluronic acid layer on at least one outer surface of the laminate.

The present invention also relates to a method for producing the above-mentioned medical substitute membrane, wherein a porous intermediate material made of a sheet-like biodegradable and absorbable material is sandwiched between two layers of collagen membrane via an adhesive. Subjecting the laminate to a first crosslinking treatment; forming a gelatin gel layer or a hyaluronic acid layer on at least one outer surface of the laminate; subjecting the laminate to a second crosslinking treatment About the method.

[0007] The medical substitute membrane of the present invention is a material derived from a living body, and has excellent biocompatibility and tissue compatibility, low antigenicity,
It has the action of promoting the expansion and proliferation of host cells, has a hemostatic effect, and has a sheet-like biodegradation absorption between two layers of collagen membrane made of collagen which is completely degraded and absorbed in vivo. A laminate having a porous intermediate material made of a conductive material via an adhesive, which is a medical alternative film having a gelatin gel layer or a hyaluronic acid layer on at least one outer surface thereof.

Hereinafter, a medical alternative film according to the present invention will be described. In this medical alternative membrane, various types of conventionally used collagen, such as neutral solubilized collagen, acid-solubilized collagen, and alkali-solubilized collagen, are used as raw materials for the two-layer collagen membrane sandwiching the porous intermediate material. Collagen or enzyme-solubilized collagen is preferable, and among these, alkali-solubilized collagen and enzyme-solubilized collagen are obtained by treating insoluble collagen with alkali or pepsin, trypsin, chymotrypsin, respectively.
It is particularly preferable because it is a treatment with an enzyme such as papain or pronase, and since these treatments remove the highly antigenic telopeptide portion in the collagen molecule and reduce the antigenicity. The origin of these collagens is not particularly limited, and generally, cows, pigs, rabbits, sheep,
Collagen obtained from skin, bones, cartilage, tendons, organs and the like of animals such as kangaroos and birds is preferred. It is preferable to use a collagen membrane made of such collagen. The thickness of the collagen membrane is preferably about 1-20 m
m, especially about 2-5 mm.

The collagen membrane in the alternative membrane of the present invention is
Preferably, in addition to the above-mentioned collagen, a natural collagen membrane of human origin, which is a biological membrane containing collagen as a main component, which is collected and purified from a human, may be a collagen membrane retaining its original membrane structure. . Because human-derived natural collagen membrane has moderate strength,
Since it is easy to handle and is the same type of protein, it has low antigenicity when applied to humans, and the capillary of the living body extends in the applied natural collagen membrane, thereby promoting regeneration of the biological membrane. In addition, after being applied to a living body, it is decomposed and absorbed by the living body, so that it does not remain in the body as a foreign substance.
A collagen film derived from human amniotic membrane, which is a medical material obtained from a living body without any problem, is particularly preferred.

The collagen membrane derived from human amniotic membrane, which can be preferably used in the medical substitute membrane of the present invention, is obtained by purifying from an integral body consisting of human fetal membrane, placenta and umbilical cord obtained immediately after parturition as a postpartum, and purified. . For example, only a fetal membrane is separated from an integral product obtained as a postpartum, the amniotic membrane is separated from the four-layered fetal membrane, and a protease inhibitor (for example, phenylmethylsulfonyl fluoride,
Wash with sonication in sterile water containing PMSF), then treat with Tris buffer containing non-ionic surfactant (eg octylphenoxypolyethoxyethanol, Triton-X, Sigma) and protease inhibitors Then, foreign substances adhering to the amniotic membrane and manually removed, further washed with sterile water, and purified by ultrasonic cleaning.

Although there has been no report that the above-mentioned CJD was transmitted from a biological material other than human brain dura mater, there is a possibility that amniotic membrane may be collected from a CJD patient. It is desirable to inactivate the causative virus of CJD by further processing for about 1 hour.

[0012] The laminate in the medical substitute membrane of the present invention described herein is used as a material for filling in a defective portion of a biological membrane, in order to secure strength enough to withstand suturing when suturing to a surgical wound.
It is a laminate in which a sheet-like porous intermediate material made of a biodegradable and absorbable material is sandwiched between two layers of collagen film and adhered with an adhesive. It is difficult to suture a laminate with only a collagen film without an intermediate material. In addition, the porous intermediate material sandwiched between the two collagen films in the present laminate is gradually decomposed and absorbed after application to a surgical wound and is replaced by a regenerated biological membrane, and thus remains as a foreign substance in the body. It is a porous sheet-like intermediate material made of a biodegradable and absorbable material that does not have any problem. As the biodegradable and absorbable material, various materials can be used as long as they are decomposed and absorbed in the living body by hydrolysis, enzymatic decomposition, etc., have no toxicity, and have a certain level of mechanical strength. Among them, polyglycolic acid (PGA), polylactic acid, a copolymer of glycolic acid and lactic acid, polydioxanone, a copolymer of glycolic acid and trimethylene carbonate, or a mixture of polyglycolic acid and polylactic acid are preferable. A porous intermediate made of glycolic acid is particularly preferred.

The thickness of the porous intermediate material made of a biodegradable and absorbable material is preferably about 10 to 50 μm, especially about 20 μm.
３０30 μm. If it exceeds about 50 μm, the collagen film will not adhere easily, and if it is less than about 10 μm, the physical properties will be weak.

The reason why the intermediate material made of the biodegradable and absorbable material is porous is that the two collagen films adhere to each other by the adhesive through the pores of the intermediate material of the porous material and are not easily separated. An integrated laminate can be obtained, and the needle passes through the hole during suturing, so that the laminate can be easily sutured to the surgical wound without tearing the laminate. is there.

The average pore diameter of the porous intermediate material is preferably about 50 to 150 μm so that the two layers of collagen membrane are closely adhered to each other through the pores of the porous intermediate material made of a sheet-like biodegradable and absorbable material. Especially about 60 to 100 μm. When the average pore diameter is less than about 50 μm, the collagen membranes are difficult to adhere to each other. When the average pore diameter exceeds about 150 μm, the collagen membrane is torn at the time of suturing, and the suturing property is reduced.
When the size of each hole is different, the average hole diameter described in the present invention is an arithmetic average when the diameter of a circle having the same area as the hole area is calculated as the hole diameter.

The adhesive for sandwiching the sheet-like porous intermediate material between the two collagen films is preferably an aqueous gelatin solution or a collagen hydrochloride solution which is also biodegradable and absorbable. . Gelatin, which is a raw material of these adhesives, is a conventionally used gelatin,
For example, collagen that is a purified gelatin of the Japan Pharmaceutical Co., Ltd. and is a raw material of the adhesive, for example, neutral solubilized collagen, acid-solubilized collagen, alkali-solubilized collagen, enzyme-solubilized collagen, etc. Such collagen may be used.

The laminate in the medical substitute membrane of the present invention has a gelatin gel layer or a hyaluronic acid layer on at least one outer surface. Since gelatin has an effect of preventing cell adhesion and proliferation in contrast to collagen, the gelatin gel layer has an adhesion to prevent the spread of cells from surrounding biological tissues in places where adhesion needs to be prevented. It can act as a barrier. In addition, hyaluronic acid has the effect of improving the stability of collagen and also has the ability to prevent adhesion. The gelatin gel layer or the hyaluronic acid layer must remain without being decomposed and absorbed for about 2 to 3 weeks after application of the medical substitute membrane of the present invention to a living body. Preferably, the layer is a gelatin layer or a hyaluronic acid layer.

Hereinafter, the method for producing the medical substitute film of the present invention will be described. In order to manufacture the laminate in the medical substitute membrane of the present invention, first, a collagen membrane is prepared from various types of collagen which have been used in the past as described in detail above. Various types of collagen, preferably neutral-solubilized collagen, acid-solubilized collagen, alkali-solubilized collagen, or enzyme-solubilized collagen, particularly preferably alkali-solubilized collagen or enzyme-solubilized collagen, are used as a raw material, and a collagen hydrochloric acid solution (about 1N, pH about 3)
Is prepared, a collagen hydrochloric acid solution layer is formed by a conventional method such as application and pouring of a collagen hydrochloric acid solution, and then dried to form a collagen film. The concentration of collagen in the collagen hydrochloric acid solution used here can be appropriately adjusted depending on the desired thickness and density of the collagen film, but is preferably 0.1 to 3% by weight, particularly 0.5 to 2% by weight. I do. The thickness of the collagen hydrochloric acid solution layer is adjusted so that the finally formed collagen film has a thickness of preferably about 1 to 20 mm, particularly about 2 to 5 mm. If the thickness of the collagen membrane is less than about 1 mm, the absorption of collagen in the living body is too early to obtain a sufficient anti-adhesion effect, and if the thickness exceeds about 20 mm,
There is a possibility that a problem of difficulty in handling may occur. This collagen membrane, after application to the surgical wound, penetration of living cells, spread,
It is preferable that the collagen solution is formed porous so that the growth can be easily performed. In order to obtain a porous collagen membrane, it is particularly preferable to use a collagen solution which is foamed by stirring.

In addition to the collagen membrane prepared as described above, a human amniotic membrane-derived collagen membrane, particularly preferably a human amniotic membrane-derived collagen membrane, can be used. Human amniotic membrane-derived collagen membrane can be obtained and purified by any method from a human fetal membrane obtained immediately after parturition and obtained as a postpartum, consisting of a placenta and an umbilical cord, for example, as described in detail above. It is preferable to use a collagen membrane derived from human amniotic membrane separated and purified by the method.

In manufacturing the medical substitute membrane of the present invention, a porous intermediate material made of the sheet-like biodegradable and absorbable material described in detail above was obtained as described above.
It is sandwiched between layers of collagen film and adhered with an adhesive to form a laminate. Lamination may be performed by any method. For example, a porous intermediate material made of a biodegradable and absorbable material in the form of a sheet is immersed in an adhesive solution, and a collagen film of one layer is laminated on both sides thereof. Air bubbles can be sufficiently removed by holding in a sandwich shape and holding, and the layers can be laminated by drying. As the adhesive, as described above,
A solution in which collagen is dissolved in hydrochloric acid (about 1N, pH about 3) or an aqueous gelatin solution is used. The concentration of the aqueous gelatin solution is, for example, about 1 to 30% by weight, but about 2.5% by weight.
Is preferred in terms of handling. The collagen concentration of the collagen hydrochloride solution used as an adhesive is preferably about 1 to 3% by weight, especially about 1% by weight.

Next, the laminate obtained above is subjected to a first crosslinking treatment. By performing the cross-linking treatment, the laminate can be adjusted so as to remain without being peeled or decomposed for about 3 to 4 weeks after application to the living body. By leaving it for about 3-4 weeks, adhesion is prevented,
The extension of the biological membrane is promoted. Further, the adhesiveness of the laminate is also improved. As a crosslinking method, gamma rays, ultraviolet rays, electron beams,
A crosslinking method using glutaraldehyde or epoxy, or a thermal dehydration crosslinking method using heat can be used.However, it is easy to control the degree of crosslinking and the thermal dehydration crosslinking that does not cause a problem with the biological effect of the crosslinking agent is performed. Is preferred. For the thermal dehydration crosslinking, the laminate obtained above is subjected to high vacuum (about -0.08 mPa or less), preferably about 105 to 15 mPa.
Below 0 ° C, especially at about 140 ° C, preferably about 12-48
Heat for hours, especially about 24 hours. Below about 105 ° C,
Sufficient adhesive strength cannot be obtained without a sufficient crosslinking reaction. If the temperature is higher than about 150 ° C., the strength of the intermediate material is reduced, and the collagen film is denatured.

A gelatin gel layer or a hyaluronic acid layer is formed on at least one outer surface of the laminate obtained as described above, that is, on both outer surfaces or one outer surface. Since gelatin has an effect of preventing cell adhesion and proliferation in contrast to collagen, the gelatin gel layer is used to prevent adhesion of cells from surrounding biological tissues at locations where adhesion needs to be prevented. It can be used as a prevention layer. In addition, hyaluronic acid has the effect of improving the stability of collagen and also has the ability to prevent adhesion.

The gelatin gel layer after crosslinking obtained by subjecting the gelatin gel layer formed here to the second crosslinking treatment described below is used as a replacement for the collagen membrane of the present alternative membrane until the surrounding tissue is regenerated. It has the role of preventing adhesion to but is gradually decomposed and absorbed after application to a living body.
Therefore, in order to allow the present gelatin gel layer to remain without being decomposed and absorbed for about 2 to 3 weeks until the biological membrane extends and regenerates from the periphery of the membrane defect and closes the membrane defect, Perform processing. In order for the gelatin gel layer after the cross-linking treatment to remain in the body for about 2 to 3 weeks after application to the living body, the gelatin gel layer is preferably prepared using an aqueous gelatin solution of about 2 to 30% by weight, particularly about 20% by weight. When an aqueous gelatin solution of about 20% by weight is used, it is preferably about 1 to 7 mm, particularly about 3 to 5 mm when wet, and preferably about 0.3 to 3 mm, particularly about 1 to 3 mm when dry. Thus, a gelatin gel layer is formed. The gelatin gel layer may be formed by any method such as coating and dipping.For example, an aqueous gelatin solution is poured into a container such as a petri dish so as to have a required thickness, and the gelatin gel layer is formed thereon as described above. The resulting laminate is left to stand and gelatin is gelatinized. When gelatin gel layers are formed on both outer surfaces, the same treatment is performed on the other surface of the laminate to form gelatin gel layers on both outer surfaces.

Next, the thus obtained laminate having the gelatin gel layers formed on both outer surfaces or one outer surface is subjected to a second crosslinking treatment. By performing the crosslinking treatment, the rate of decomposition and absorption of the gelatin gel layer is controlled. As a crosslinking method, thermal dehydration crosslinking is also preferable for the same reason as described above. In order to carry out thermal dehydration crosslinking and leave the gelatin gel layer for about 2 to 3 weeks after application to the living body, the laminate on which the gelatin gel layer has been formed is subjected to high vacuum (about -0.08 mPa or less). , Preferably about 105 to 15
Below 0 ° C, especially at about 120 ° C, preferably about 12-48
It is subjected to a thermal dehydration crosslinking treatment for a period of time, especially about 24 hours. About 105
If the temperature is lower than 0 ° C, the crosslinking reaction does not sufficiently occur, and if the temperature is higher than about 150 ° C, the collagen membrane is denatured.

When the hyaluronic acid layer is formed, the laminate obtained as described above using an aqueous solution of sodium hyaluronate of preferably about 0.5 to 2.0 mg / ml, particularly about 1.0 mg / ml, is used. A sodium hyaluronate aqueous solution layer is formed on at least one of the outer surfaces, that is, both outer surfaces or one outer surface, by a method such as coating or dipping, and the aqueous solution layer is air-dried to form a hyaluronic acid layer. The aqueous solution layer of sodium hyaluronate can remain without decomposing and absorbing the hyaluronic acid layer for about 2 to 3 weeks until the biological membrane extends from the periphery of the membrane defect and regenerates and closes the defect part of the membrane. So that it is preferably about 0.
5 to 4.0 mm, especially about 2 mm, preferably about 0.
It is formed to a thickness of 1 to 2.0 mm, especially about 1.0 mm (in the case of an aqueous solution of about 1.0 mg / ml). In order to fix hyaluronic acid on the surface of the laminate and form a hyaluronic acid layer, a second crosslinking treatment is further performed. In the case of hyaluronic acid, crosslinking treatment with water-soluble carbodiimide (WSC) is preferred. preferable. In this case, by mixing WSC in advance with the aqueous solution of sodium hyaluronate and applying it to the laminate together with sodium hyaluronate,
It is preferable to crosslink the carboxyl group of collagen and the amino group of hyaluronic acid. The concentration of WSC contained in the aqueous solution of sodium hyaluronate is preferably about 5
-20 mg / ml, especially about 8-15 mg / ml. An aqueous solution containing the sodium hyaluronate and WSC is prepared, thoroughly stirred, applied to at least one outer surface of the laminate so as to have a thickness of preferably about 2 mm, air-dried, and dried. Form a layer.

The medical substitute membrane of the present invention obtained as described above is used to compensate for the membrane defect after various surgical operations, thereby preventing the adhesion between the organ and the surrounding tissue at the membrane defect. It can be used as a biological replacement membrane. In the alternative membrane of the present invention, a gelatin gel layer or a hyaluronic acid layer is formed on one or both surfaces so that the cross-linked gelatin gel layer or the hyaluronic acid layer faces the side in contact with the peripheral tissue in which adhesion is required to be prevented. The formed alternative membrane of the present invention is used. When using this medical replacement membrane as a replacement membrane for the pericardium, use a replacement membrane with a gelatin gel layer or a hyaluronic acid layer formed on both surfaces, and use one side when using it as a replacement membrane for the pleura, peritoneum or serosa. An alternative membrane having a gelatin gel layer or a hyaluronic acid layer formed on the surface is used so that the gelatin gel layer or the hyaluronic acid layer faces the side in contact with the surrounding tissue. When used as an alternative membrane for the brain dura, any of the alternative membranes having a gelatin gel layer or a hyaluronic acid layer formed on both surfaces or one surface can be used. When an alternative membrane having a gelatin gel layer or a hyaluronic acid layer formed on one surface is used, the gelatin gel layer or the hyaluronic acid layer is used such that the gelatin gel layer or the hyaluronic acid layer faces the side in contact with the brain parenchymal tissue.

As described above, the medical substitute membrane of the present invention as a material for filling a defective portion of a biological membrane can be used as a substitute membrane for brain dura, pericardium, pleura, peritoneum or serosa. When this alternative membrane is applied to a surgical wound, the biological membrane such as the dura mater, pericardium, pleura, peritoneum, or serosa remaining around the surgical wound will be replaced with the collagen membrane of the alternative membrane from the point of contact with the alternative membrane. While extending and regenerating as a scaffold for regeneration, where the biological tissue contacts the gelatin gel layer or the hyaluronic acid layer,
Adhesion is prevented because cell invasion and extension are prevented,
Eventually, the defective part is closed by the regenerated biological membrane,
This substitute membrane is decomposed and absorbed by the living body and completely disappears.

[0028]

The present invention is not limited to the following examples, which describe the medical substitute membrane and the method for producing the same in detail.

Preparation Example 1 Purification of Human Amniotic Membrane The fetal membrane was separated from the monolith obtained as a postpartum, the amniotic membrane was separated from the fetal membrane, and a protease inhibitor (phenylmethylsulfonyl fluoride, PMSF) 0.35 mg / 1% octylphenoxypolyethoxyethanol (Triton X-100, Sigma) in a Tris buffer solution (PMSF) by sonication (1 kw transmitter) with sterile water containing l. 0.35 mg / l further), treated at room temperature for 24 hours, manually removed foreign substances still adhering to the amniotic membrane, washed with sterile water at room temperature for 48 hours, and sterilized water at room temperature. Sonication for 2 hours (1kw
The amniotic membrane was treated with a 1N aqueous solution of NaOH at room temperature for 1 hour, washed with sterile water at room temperature for 1 hour, and dried to obtain a human amniotic membrane-derived collagen membrane. In the collagen membrane obtained by this method,
The cells were completely removed.

Preparation Example 2 Preparation of Laminate The collagen membrane derived from human amniotic membrane prepared and purified and stored in Preparation Example 1 was immersed in sterilized water. A sheet-like PGA mesh (size: 10 cm × 15 cm, pore size: 80 μm, thickness: 50 μm, manufactured by Mitsui Toatsu Chemicals, Inc.) as a sheet-like biodegradable absorbent material is immersed in a 2.5% by weight gelatin aqueous solution. Then, bubbles attached to the PGA mesh were removed. Next, the collagen membrane was overlaid on a PGA mesh. About 7 ml of a 2.5% gelatin solution is added to the other surface of the PGA mesh on which the present collagen membrane is not laminated, and another human-derived collagen membrane derived from human amniotic membrane is laminated thereon to form a sandwich. It was left still at 4 ° C. for 1 hour. The laminate was air-dried and then dried under reduced pressure in a desiccator. Next, the laminate obtained above was placed in a vacuum oven (YAMA).
Using TO, model: DP43) under high vacuum (about -0.08mP
a)), subjected to thermal dehydration crosslinking treatment by leaving still at 105 ° C., 120 ° C., 130 ° C., 140 ° C. or 150 ° C. for 12 hours or 24 hours, and then stored in a dry state at room temperature.

Test Example 1: Adhesion test of laminate The adhesion test of the laminate prepared in Preparation Example 2 and subjected to the thermal dehydration crosslinking treatment was performed. The laminate subjected to the thermal dehydration and crosslinking treatment in Preparation Example 2 was cut into strips (1 × 2.5 cm), immersed in 20 ml of physiological saline at 37 ° C., and stored. Was pinched with tweezers and pulled to observe whether the film was broken or peeled off. Observation of the process until the collagen film derived from the amniotic membrane of the laminated body immersed in physiological saline and subjected to thermal dehydration cross-linking under each condition was peeled off from the PGA mesh by the external force, and the progress until the collagen film was spontaneously peeled off while standing Table 1 shows the results.

[0032]

[Table 1]

In the laminate subjected to thermal dehydration crosslinking at 105 ° C. for 12 hours under a high vacuum, all the examples peeled off on the second day when an external force was applied. In the case of standing, the amniotic membrane-derived collagen membrane was spontaneously separated from the PGA mesh on the fifth day in all cases.
In the laminate subjected to thermal dehydration crosslinking at 120 ° C. under high vacuum, the collagen film and the PGA mesh were separated in one of four cases on the second day when an external force was applied. On the seventh day, the collagen membrane was detached in all cases. On the 8th day,
In all cases, the collagen film and the PGA mesh were spontaneously separated. In the laminate subjected to thermal dehydration crosslinking at 130 ° C. under high vacuum, the collagen film was peeled off in three out of four cases on the fourth day when an external force was applied. On the sixth day, peeling occurred in four out of four cases. When left standing, the collagen membrane and the PGA mesh were completely peeled off on the 18th day in 4 out of 4 cases.

In the laminate subjected to thermal dehydration crosslinking at 140 ° C. under high vacuum, the collagen film was peeled off in one of four cases on the fourth day when an external force was applied. In all cases, the collagen membrane was PG
It peeled off from the A mesh 12 days later. When allowed to stand, the collagen membrane and PG were observed in all cases after 20 days.
The A mesh spontaneously peeled off. In the laminate subjected to thermal dehydration crosslinking at 150 ° C. under high vacuum, the collagen film was peeled off in four out of four cases on the ninth day when an external force was applied. If left unattended,
It was 23 days after the collagen membrane was detached from the PGA mesh in all cases.

From the above results, it was confirmed that the adhesiveness between the collagen membrane and the PGA mesh was improved in proportion to the thermal dehydration crosslinking temperature. However, when the laminate dehydrated and crosslinked at 150 ° C. is pulled with tweezers, the PGA of that portion is pulled.
The mesh was observed to tear, suggesting that if the crosslinking temperature was too high, the strength of the PGA mesh would decrease. From these results, it was recognized that, among the temperature conditions tested, 140 ° C. was the most appropriate thermal dehydration crosslinking temperature for improving the adhesive strength.

Example 1 Preparation of Alternative Medical Film Having Gelatin Gel Layer An aqueous gelatin solution of about 20% by weight was prepared. In Preparation Example 2, under high vacuum (approximately −0.08 mPa or less) at 140 ° C.
A gelatin gel layer was formed on one outer surface of the laminate prepared by subjecting the laminate prepared by subjecting to a thermal dehydration crosslinking treatment to a dry thickness of about 1 to 2 mm. This is subjected to high vacuum (about -0.08 mPa or less) at 120 ° C. for 24 hours,
By subjecting it to a thermal dehydration crosslinking treatment, a medical substitute membrane of the present invention was obtained.

Example 2 Preparation of Alternative Medical Film Having Hyaluronic Acid Layer An aqueous solution containing sodium hyaluronate 1.0 mg / ml and water-soluble carbodiimide (WSC) 10 mg / ml (pH 5.
0) was prepared and stirred well at room temperature. In Preparation Example 2, the reaction was carried out at 140 ° C under high vacuum (about -0.08 mPa or less).
The aqueous solution was applied to both outer surfaces of the laminate prepared by subjecting it to a thermal dehydration crosslinking treatment for 4 hours to form an aqueous sodium hyaluronate layer having a thickness of about 2 mm. This was air-dried and then subjected to a sterilization treatment to obtain a medical substitute membrane of the present invention.

Test Example 2: Anti-adhesion effect A book having a gelatin gel layer on one outer surface in the same manner as in Example 1 except that the gelatin gel layer was formed so that the thickness when dried was about 3 mm. The medical substitute film of the present invention was prepared, and its adhesion preventing effect was examined. Twenty-four female New Zealand white rabbits were subjected to a midline abdominal incision under anesthesia, and the serosa was excised on both sides of the cecum in a size of 2 × 3 cm. In addition, the peritoneum of the abdominal wall facing the serosal deficient part was 3 ×
The excision was performed at a size of 4 cm. In the control group (12 animals), the abdomen was closed as it was, and in the test group, the surface having the gelatin gel layer of the medical substitute membrane of the present invention was directed to the abdominal cavity, and sutured and fixed to the serosal defect of the cecum, and the abdomen closed. Rabbits were sacrificed 2 or 6 weeks after the operation, and the degree of adhesion was observed macroscopically and histologically.

No deaths of any rabbits were observed during the post-operative breeding period. In the control group, 1 out of 12
In one animal, strong adhesion was observed at the resected site. In the group to which the medical substitute membrane of the present invention was applied, adhesion was not observed in 10 out of 12 animals, and mild adhesion was observed in 2 animals. Histologically, two weeks after the operation, in the rabbit abdominal wall to which the medical substitute membrane of the present invention was applied, the peritoneum was normally regenerated and healed. The site was covered, and a slight gelatin gel layer remained.
At 6 weeks after the operation, the replacement membrane had almost disappeared, and the surface of the serostomy site was covered with the serosa. From the above results, it was confirmed that the medical substitute membrane of the present invention having a gelatin gel layer was useful for preventing adhesion. It is considered that the gelatin gel layer gradually turned into a sol after the replacement membrane was applied to a living body, so that it was difficult for the living tissue to enter the replacement membrane, and this adhesion preventing effect was obtained.

Test Example 3: Effect as a substitute pericardium A medical substitute film of the present invention having a hyaluronic acid layer on both outer surfaces prepared in Example 2 and a stretched polytetrafluoroethylene film material (EPTFE) as a control
Was cut into 2 x 2 cm squares and used as a pericardial patch in the following experiments. The pericardium of 10 mongrel dogs and beagle dogs was cut into a square of 2 × 2 cm, and the cut portion was filled with the above-mentioned patch for medical replacement membrane (5 dogs) or EPTFE (5 dogs). The four corners were single-knot sutured using 4-0 or 6-0 prolene sutures. 37-102 after surgery
On the day, the dogs were sacrificed and the degree of adhesion of the post-surgical repair site was observed macroscopically and histologically using hematoxylin and eosin staining. In addition, the pericardium and heart around the pericardial replacement site were observed with an electron microscope.

No deaths of any dogs were observed during the post-operative breeding period. In addition, in all the pericardial patches, the suturing property was good, and at the time of suturing, the collagen membrane derived from amniotic membrane of the medical substitute membrane of the present invention did not tear or wrinkled due to blood. In a dog to which a pericardial patch using the medical replacement membrane of the present invention was applied, 5
In three of the heads, weak and moderate adhesions to the heart were noted.
The surface of the pericardial replacement site in dogs that did not show adhesions was covered with a thin capsule, where neovascularization was observed. Histological observation revealed that the regenerated connective tissue had formed a membrane of a certain thickness, and a layer of mesothelial cells had regenerated on the surface of the connective tissue membrane. Observation of the complemented site by electron microscopy confirmed that a layer of mesothelial cells with microvilli covered the surface of the connective tissue membrane rich in capillaries, and similar findings were found at the central site supplemented. And tissue regeneration was confirmed to extend to the entire patch. The basement membrane was also normal. In dogs to which the EPTFE patch was applied, weak / medium adhesion to the heart was also observed in 3 out of 5 dogs. The interface between the pericardial replacement site and the pericardium was cloudy, and such a finding was not observed in the pericardial patch using the alternative membrane of the present invention. Histological observation showed that the EPTFE patch was
It was covered by a coat, and only a small part of the coat showed mesothelial cells, and most of the coat had exposed connective tissue. No neovascularization was observed in the capsule. At places where cloudiness was observed with the naked eye, fatty degeneration of myocardial tissue was remarkably observed. EPT
Observation of the FE patch-supplemented site with an electron microscope revealed that no cell engraftment was observed on the patch surface, no microvilli were observed, and only a multiple structure of connective tissue was observed. No skin cells were observed and the connective tissue was exposed. In addition, in the cloudy spot on the interface between the replacement site and the pericardium, there was no portion that would be covered with microvilli.

From the above findings, the patch using the medical substitute membrane of the present invention exhibits an anti-adhesion effect comparable to the EPTFE patch, and after replacement, is replaced by the regenerated pericardium, and the regenerated mesothelial cells, While the basement membrane and lower connective tissue are also close to normal, the EPTFE patch only covers a thin coat of connective tissue and does not show mesothelial cell layer regeneration,
It was found that fat degeneration occurred at the contact point between the PTFE patch and myocardial tissue. From these points, the medical substitute membrane of the present invention has an anti-adhesion effect comparable to EPTFE and has the EP
It was found to be better than TFE.

[0043]

The medical substitute membrane of the present invention can be stably supplied without any ethical problems, and can be sutured to a surgical wound as a material for filling a defective part of a biological membrane or an adhesion preventing material. Also, after suturing, it remains for a period until the biomembrane regenerates and exhibits an anti-adhesion effect, but is gradually decomposed and absorbed, so it can be used safely without remaining in the living tissue for a long period of time to cause inflammation. can do.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Toru Tsuda 3-17-8, 304 Ogaya, Otsu-shi, Shiga Prefecture, Japan (72) Inventor Masami Teramachi 68, Honcho, Himeji-shi, Hyogo Pref. Inventor Yukinobu Takimoto 8-8 Amenity Futagaoka 307, 8-8 Tokiwayamashita-cho, Ukyo-ku, Kyoto-shi, Kyoto (72) Inventor Tatsuo Nakamura 158-2, Senbon-Nishiiri Gobancho, Ninnaji-kaido, Kamigyo-ku, Kyoto, Kyoto Prefecture 1109

Claims (8)

[Claims] 1. A laminate having a sheet-like porous intermediate material made of a biodegradable and bioabsorbable material via an adhesive between two layers of a collagen membrane, wherein at least one of the laminates has an outer surface. A medical alternative film having a gelatin gel layer or a hyaluronic acid layer on the surface. 2. The medical substitute membrane according to claim 1, wherein the collagen membrane is a human-derived natural collagen membrane. 3. The medical alternative membrane according to claim 2, wherein the human-derived natural collagen membrane is a human amniotic membrane-derived collagen membrane. 4. A collagen membrane comprising a neutral solubilized collagen, an acid solubilized collagen, an alkali solubilized collagen,
2. The medical alternative membrane according to claim 1, wherein the membrane is made of enzyme-solubilized collagen. 5. The method according to claim 1, wherein the porous intermediate material is polyglycolic acid (P
GA), polylactic acid, a copolymer of glycolic acid and lactic acid,
The medical substitute membrane according to claim 1, comprising a material selected from polydioxanone, a copolymer of glycolic acid and trimethylene carbonate, and a mixture of polyglycolic acid and polylactic acid. 6. The gelatin gel layer or the hyaluronic acid layer,
The medical substitute membrane according to claim 1, which is a crosslinked gelatin gel layer or hyaluronic acid layer. 7. The medical substitute membrane according to claim 6, wherein the crosslinked gelatin gel layer or hyaluronic acid layer is crosslinked to such an extent that it can remain in vivo for 2 to 3 weeks. 8. The method for producing a medical substitute membrane according to claim 6, wherein a porous intermediate material made of a sheet-like biodegradable and absorbable material is sandwiched between two collagen films via an adhesive. Forming a laminate; subjecting the laminate to a first crosslinking treatment;
Forming a gelatin gel layer or a hyaluronic acid layer on at least one outer surface of the laminate; and subjecting the laminate to a second crosslinking treatment.