JP2000093497A - Collagen film for medical treatment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a collagen film for medical treatment which solves all of the suture strength, sufficient bioadaptability and the lack of tissue regeneration acceleration performance which are the drawbacks associated with the conventional collagen films for medical treatment. SOLUTION: This collagen film for medical treatment is formed by covering at least one surface of a nonwoven fabric layer consisting of crosslinked collagen fibers with a collagen coat. The coat layer promotes the tissue regeneration of the transplanted defect sections, etc., by exhibiting the properties, such as a hemostatic effect and adhesion, propagation and expansion of cells which are the advantages of collagen. The nonwoven fabric layer exists as the skeleton for supplementation, prosthesis and seal of the defect section, etc., until the tissue regeneration of the in-vivo defect sections, etc., is completed. The nonwoven fabric layer is completely decomposed and-absorbed like the coat layer after maintaining the film strength for the specified period after the suture and fixation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a collagen film for medical use. Specifically, it is used as a substitute membrane for living tissue, for filling in a defective part or a cut surface of various organs, such as pleura, pericardium, cerebral dura, and serosa, and for prosthesis, and is particularly sutureable. Also, the present invention relates to a medical collagen membrane which has good biocompatibility and can induce promotion of tissue regeneration.

[0002]

2. Description of the Related Art In a general surgical operation, an affected part is often resected and a damaged part is repaired. In particular, when various organs such as a lung, a heart, a liver, and a brain are targeted, the operation is performed. Unless the membrane covering the tissue on the cut surface or the defective part is supplemented or prosthetic, the fundamental function of the organ is often impaired. If these actions are performed incompletely,
Even if they die from organ dysfunction or escape from a life-threatening crisis, the prognosis tends to be very poor. For the purpose of preventing such dysfunction of organs and tissues, membranes covering the organs or tissues have been developed using various materials.

At present, typical substitute membranes for in vivo membrane-like tissues such as pleura, pericardium, cerebral dura, and serosa include those using synthetic fibers, those using animal and human tissues, and various types. Materials using biological materials such as gelatin and collagen are known. Alternative films using these various materials have excellent advantages due to the properties derived from each material, but also have conflicting disadvantages, and there are still alternative films that satisfy all conditions. Absent.

[0004] For example, an alternative membrane using synthetic fibers has excellent mechanical strength to the extent that it can be sutured and fixed to a target site irrespective of bioabsorbable or nonabsorbable materials. On the other hand, since it is a synthetic product, it is recognized as a foreign substance in the body, and a satisfactory result is not obtained in terms of biocompatibility, such as frequent encapsulation. In addition, those using animal or human tissues have almost satisfactory mechanical strength and somewhat good biocompatibility, but they are ultimately heterogeneous tissues, and the transplanted side is completely rejected. It cannot be denied that calcification has been reported. In addition, the use of human cadaver tissue is a source of ethical and quantitative problems, and in recent years CJD (Creutzfeldt-JacobD)
isease) has been reported and is a social problem. on the other hand,
From the viewpoint of medical safety and biocompatibility, it is most ideal to use self-tissues such as the own fascia, but this is not always possible from the target patient's own situation. In addition, even if it is possible to collect it, it is impossible to collect a large amount even if it can be collected, and there is a big problem that the patient himself is extremely distressed.

[0005] Under such circumstances, various attempts have been made to commercialize a film-like material having a performance close to that of a self-tissue using various biological materials such as gelatin, collagen, and hyaluronic acid. For example, Japanese Unexamined Patent Publication No. Hei 7-502431 is an example of using hyaluronic acid.However, since the present invention is mainly based on synthetic or semi-synthetic route of hyaluronic acid, it is a pure bio-based material. It was difficult to say that it was a material, and was not sufficient in terms of biocompatibility in tissue regeneration.

[0006] Further, as examples utilizing animal or human tissues, Japanese Patent Application Laid-Open No. 9-502371, Japanese Patent Application Laid-Open No. 9-122227, Japanese Patent Application Laid-Open No. Hei 7-116242 and the like can be mentioned. However, when using a human organization, the above-mentioned CJD
In addition, the problem of infectivity cannot be completely denied, and a membrane obtained by purifying human tissue has not been obtained with sufficient strength to endure sutures.
Among these patents, only Japanese Patent Application Laid-Open No. Hei 7-116242 describes that suturing was possible in surgical operations, but this was achieved by using synthetic fibers such as polyglycolic acid. However, the medical disadvantages described above when using synthetic molded articles have not been eliminated.

On the other hand, various attempts have been made to combine a synthetic molded article with collagen derived from animals to improve the film strength as a medical material and the strength of a fibrous material. These techniques include JP-A-1-131666, JP-B-7-87850, JP-A-7-178131, and
There are a large number of publications such as -113384. However, no matter how good the suture strength can be, they cannot escape from medical disadvantages such as foreign body reaction and skin encapsulation during implantation in the body, as long as they use synthetic molded products as described above. Therefore, it is difficult to become an ideal medical substitute membrane. In addition, as similar techniques, Japanese Patent Application Laid-Open No. Hei 7-94689 and Japanese Patent Application Laid-Open No. Hei 6-292716 have been disclosed, but suture strength,
It is hard to say that all of the three aspects of biocompatibility and induction of tissue regeneration are sufficiently satisfied.

[0008] There is a medical membrane using only animal-derived collagen in contrast to the one using such a synthetic molded article. For example, JP-T-59-501319, JP-B-61-347
No. 98, JP-A-5-105624, JP-A-5-253285, JP-T-7-501004, JP-T-7-509143, JP-A-8-52204, and JP-A-9- No. 31334, Japanese Unexamined Patent Publication No.
Japanese Patent No. 304240, Japanese Patent No. 2607266, and the like are known. Among these, Japanese Patent Application Laid-Open No. Hei 7-509143 discloses that a suture strength has been achieved in the dental area, but the surface of collagen using a cross-linking agent or the like directly contacts the living body. Therefore, there were problems in biocompatibility, induction of tissue regeneration promotion, and the like.

[0009] Further, Japanese Patent No. 2607266 introduces a technique of a medical material that can be sewn qualitatively when implanted into an animal. This patent shows that by using an in-vivo substance such as ketose as a cross-linking agent, safety to a living body is emphasized, and that biocompatibility is good even in an implantation experiment. However, since the surface of the film-like material that comes into contact with the living body uses a crosslinking agent, there is a problem from the viewpoint of biocompatibility and induction of promotion of tissue regeneration. Further, Japanese Patent No. 2610471 discloses a technique for obtaining a porous sponge of collagen. The collagen porous sponge obtained by this patent is good in terms of biocompatibility and inducing the promotion of tissue regeneration, but cannot be sutured and fixed due to the strength of the sponge. Further, in addition to these patents, Japanese Patent Application Laid-Open No. 4-500954,
JP-B-503371, JP-B 5-49303, JP-B 8-11121
And Japanese Patent Publication No. 4-21496, however, it is difficult to say that all of them are sufficiently satisfied in the three points of suture strength, biocompatibility, and induction of tissue regeneration.

[0010] Similarly, as a technique for improving the strength of a film-like material by using only animal-derived collagen and processing it into a nonwoven fabric, Japanese Patent Application Laid-Open No. 50-141190,
There are JP-B-51-42234 and JP-B-62-34880. However, these were all developed for the purpose of wound dressings and skin substitute membranes, and there were no considerations for applications premised on suturing. Despite the implementation,
It cannot withstand surgical procedures that require suturing. In addition, it is treated with a chemical such as a cross-linking agent for the purpose of imparting water resistance, and the surface to be treated is in direct contact with a living body, and lacks consideration for biocompatibility and induction of tissue regeneration.

[0011]

All of the medical membranes using these collagens have a problem that the collagen membrane lacks suture strength, sufficient biocompatibility and tissue regeneration promoting performance. I was An object of the present invention is to provide a medical collagen membrane that has solved the drawbacks of the conventional medical collagen membrane, such as insufficient suture strength, sufficient biocompatibility, and insufficient tissue regeneration promoting performance. The present inventors have conducted intensive studies to solve the drawbacks of the conventional medical collagen membrane, and as a result, formed a collagen membrane having a laminated structure composed of a nonwoven fabric layer composed of cross-linked collagen fibers and a collagen coating. By doing so, the inventors have found that the object of the present invention is achieved, and reached the present invention.

[0012]

That is, the present invention is a medical collagen membrane in which at least one surface of a nonwoven fabric layer composed of crosslinked collagen fibers is covered with a collagen coating. Further, the present invention provides the medical collagen membrane, wherein the collagen fiber constituting the nonwoven fabric has a cross-sectional diameter of 10%.
It is a collagen film for medical use having a size of ~ 1000 µm. Further, the present invention is the medical collagen membrane, wherein the collagen coating comprises a porous collagen.

Furthermore, the present invention is the medical collagen membrane, wherein the nonwoven fabric layer is a crosslinked collagen fiber and the fiber space is filled with collagen. The present invention also relates to the medical collagen membrane, wherein the collagen is at least one selected from enzyme-solubilized collagen, acid-solubilized collagen, alkali-solubilized collagen, and neutral solubilized collagen. is there. Further, the present invention is a medical collagen film obtained by freeze-drying a porous collagen film in the medical collagen film. Furthermore, the present invention relates to the medical collagen membrane, wherein
Collar filled in a gen coating and / or nonwoven layer
It is a collagen film for medical use which is made by cross-linking Gen.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The collagen film for medical use of the present invention comprises:
It is produced using collagen, which is a biological material, as a main raw material, and has a laminated structure of a nonwoven fabric layer and a coating layer. The collagen film for medical use is a laminated film in which a nonwoven fabric layer is sandwiched in several places in a laminated structure with a coating layer, and a laminated structure of 2 to 8 layers in which at least one surface of the laminated film is a coating layer is preferable. The nonwoven fabric layer contributes to the strength of the medical collagen membrane that can be sutured, and the coating layer contributes to the improvement of biocompatibility and the performance of promoting tissue regeneration.

That is, since the medical collagen membrane itself is entirely composed of collagen, which is a bio-derived material, not only is the biocompatibility excellent, but also it is gradually decomposed and absorbed in the transplanted living body, and the final Is all disassembled
Absorbed. In the process of this decomposition and absorption, the coating layer mainly consists of the hemostatic action, cell adhesion and
It exhibits properties such as proliferation and extension, and promotes tissue regeneration at a transplanted defect site or the like. In addition, the nonwoven fabric layer is used as a skeleton for filling, prosthesis, and sealing the defective site until the tissue regeneration of the in vivo defective site is completed, and after maintaining the film strength for a certain period after the suture fixation, All are decomposed and absorbed like the coating layer. Since the nonwoven fabric layer is also made of collagen, it has not only a role of maintaining the strength but also a considerable amount of properties such as hemostasis, cell adhesion, proliferation and extension, like the coating layer.

The collagen used in the present invention includes:
Examples include enzyme-solubilized collagen, acid-solubilized collagen, alkali-solubilized collagen, and neutral solubilized collagen. These solubilized collagens are, for example, those solubilized by proteolytic enzymes such as pepsin and trypsin or solubilized by an acid, alkali, or neutral agent. What is usually called atelocollagen, which has been subjected to removal treatment of the base telopeptide, and is suitable for medical use is preferable. These solubilized collagens are
Pigs, birds, fish, rabbits, sheep, rats, humans, etc.
From the animal species such as tendon, bone, cartilage, organs etc.
No. 33, JP-B-43-25839 and JP-B-43-27513 can be obtained by various extraction techniques.
The type of collagen is not limited to any of the types that can be classified, such as type I and type ■, but type I collagen is particularly preferable from the viewpoint of handling. Water is suitable as a solvent for solubilizing collagen.

The collagen fibers constituting the nonwoven fabric layer are formed by continuously spinning an aqueous collagen solution from a nozzle and receiving the collagen fibers in a container containing a solvent insoluble in collagen or on a conveyor belt. Get things. Next, the fibrous molded product is dried under reduced pressure, air dried,
It is dried by a method such as low-temperature drying or blast drying. In any drying method, in order to prevent the denaturation of collagen, it is important to perform the drying at a temperature lower than the denaturation temperature of the collagen to be used, and the temperature depends on the type of the collagen to be used. Or less. Particularly, low-temperature drying or reduced-pressure drying is preferable. On the other hand, a short staple-like fiber can be obtained in either continuous or discontinuous spinning by cutting the yarn obtained after discontinuous spinning by intermittent discharge or ordinary continuous spinning. In a state where these are uniformly dispersed in a container of an appropriate size,
The fibrous molded product can be obtained by drying by a method such as drying under reduced pressure and natural drying.

The concentration of the aqueous collagen solution used for spinning varies depending on the type of collagen used.
It is 0.1 to 20% by weight, preferably 1 to 10% by weight. Examples of the coagulation bath for wet spinning include an aqueous solution of an inorganic salt, an organic solvent dissolving an inorganic salt, alcohols, and ketones. For example, examples of the aqueous solution of inorganic salts include sodium sulfate, sodium chloride, ammonium sulfate, calcium chloride, and magnesium chloride, and particularly preferred are sodium chloride, sodium sulfate, and ammonium sulfate. An organic solvent in which these inorganic salts are dissolved / dispersed in alcohol or acetone may be used, and an ethanol-dissolved / dispersed solution of sodium chloride in ethanol is particularly preferable. Examples of alcohols include methanol, ethanol, isopropanol, and amyl alcohol, and ethanol is preferred for medical use. Further, as the ketones, acetone, methyl ethyl ketone and the like are preferable.
Although the method for producing the collagen nonwoven fabric of the present invention has been described centering on wet spinning, it can also be produced by dry spinning.

The collagen fiber constituting the nonwoven fabric of the present invention has a fiber cross-sectional diameter of 10 to 1000 μm, preferably 20 to 500 μm.
m, more preferably 30 to 200 μm. If the fiber cross-sectional diameter is less than 10 μm, the strength of the medical collagen film tends to be low, and if it exceeds 1000 μm, film formation tends to be difficult. Next, the collagen fibers constituting the nonwoven fabric are subjected to a crosslinking treatment. By the cross-linking treatment, the collagen nonwoven fabric has improved physical strength at the time of moistening, can sufficiently secure the strength required for suturing, and does not have time to be decomposed and absorbed when implanted in a living body. The delay can be drastically reduced as compared with the case of crosslinking. The cross-linked nonwoven fabric compensates or prostheses for the defect in the living body, prevents dysfunction of organs and tissues, etc. due to the defect, and furthermore, it is necessary for the body to repair the wound surface and complete the regeneration of the tissue. It can remain in the living body while maintaining the film strength.

The crosslinking method is roughly classified into a physical crosslinking method and a chemical crosslinking method. Examples of physical crosslinking methods include γ-rays, ultraviolet rays, electron beams, plasma, and thermal dehydration crosslinking. On the other hand, as a typical example of the chemical crosslinking method, fibers are crosslinked by treatment with aldehydes such as dialdehyde and polyaldehyde, epoxies, carbodiimides, isocyanates, tannin and chromium. Among them, aldehydes include formalin, glutaraldehyde, acid aldehyde, glyoxal, malondialdehyde, succindialdehyde, phthalaldehyde, dialdehyde starch, polyacrolein, polymethacrolein, and epoxies. Glycerol diglycidyl ether, sorbitol diglycidyl ether, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether and the like. Further, examples of the carbodiimides include water-soluble carbodiimides. Examples of the isocyanates include hexamethylene diisocyanate and tolylene diisocyanate.

The nonwoven fabric layer is formed by randomly entangled continuous collagen fibers. If necessary, the gap between the entangled collagen fibers is filled with collagen to increase the strength of the nonwoven fabric layer. At the same time, the adhesive strength between the nonwoven fabric layer and the coating layer can be increased. The collagen fiber constituting the nonwoven fabric layer can be formed into a poorly water-soluble collagen fiber by subjecting it to a chemical cross-linking method, for example, cross-linking using aldehydes such as glutaraldehyde. One or both sides of the non-woven fabric layer composed of the collagen fibers cross-linked in this way are coated with a coating layer to be integrated, and finally subjected to thermal dehydration cross-linking to induce suture strength, biocompatibility, and tissue regeneration. It is possible to obtain a collagen membrane having excellent quality. When the collagen fiber gap is filled with collagen in the nonwoven fabric layer, it is preferable that the collagen-filled layer is also thermally dehydrated and crosslinked like the coating layer. However, this is merely an example, and there is no problem even if all the layers are treated by thermal dehydration crosslinking, for example, and γ-ray irradiation may be performed for both sterilization and crosslinking. That is, the cross-linking treatment in the manufacturing process of the present medical collagen membrane is necessary to improve the physical strength of the medical collagen membrane when wet, and is necessary to secure the strength required for suturing. There is no particular limitation on the order and the like.

Further, by compressing the collagen fibrous molded product in order to obtain a higher suture strength, the fiber density of the nonwoven fabric layer is increased, and a film-shaped molded product having higher strength can be obtained. Compression can be carried out with a general-purpose press, but since it is intended for medical use, it is aseptically packed with a sufficiently strong sterilized packaging material, such as an aluminum pack or high-strength flexible resin packaging material. It is preferable that the compression is performed in the compressed state. At this time, the pressure for compressing the nonwoven fabric layer is preferably 10 to 1000 kgf / cm ² . The non-woven fabric layer is compressed as much as possible and the bulk density is increased by making it thinner, and by laminating this into a multilayer structure, it becomes a superior collagen film for medical use with higher suture strength. Become.

The collagen film for medical use of the present invention has a structure in which at least one surface of the collagen nonwoven fabric layer is covered with a collagen coating layer. When the collagen coating layer is a porous collagen, the tissue cells in the living body can extend to the pores and the medical collagen membrane can be firmly bonded to the living tissue cells, which is preferable. For example, a porous collagen film is formed by casting a solubilized collagen aqueous solution in a flat container, sufficiently freezing it in a general-purpose freezer, and then drying it with a freeze dryer to form a uniform porous collagen layer. can get. At this time, the pore size of the fine pores formed in the porous collagen layer can be adjusted by the concentration of collagen, its solvent, freezing temperature, freezing time, and the like. The thickness of the porous collagen layer is determined by the decomposition / absorption time when implanted in a living body,
It can be adjusted arbitrarily in consideration of the influence on the induction of tissue regeneration.

The porous collagen layer is prepared by casting an aqueous collagen solution having a collagen concentration of 0.5 to 5 wt%, preferably 1 to 3 wt%, in a flat container and freezing at a temperature of -196 to-in a freezer or a deep freezer. 10 ° C., preferably −80
After freezing at ~ -10 ° C, a porous collagen layer is obtained by drying in a freeze dryer. The thickness of the porous collagen is preferably 1 to 20 mm. The collagen film for medical use is formed by bonding the porous collagen layer and the nonwoven fabric layer thus formed to each other by applying pressure using a solubilized collagen or the like, or by bonding the nonwoven fabric layer to an aqueous solution of the solubilized collagen before freezing. It can be manufactured by a method such as integration by dipping and freeze-drying. The total amount and thickness of the collagen in the porous collagen layer are generally about 1 so as not to hinder the repair of wounds such as the target defect, the cut surface, and the induction of tissue regeneration.
It is desirable that the porous collagen film remains in the body for about 4 weeks.

The medical collagen membrane comprising the nonwoven fabric layer and the porous collagen layer thus obtained can be further compressed by applying pressure. Further, after the porous collagen layer alone or the nonwoven fabric layer alone is compressed, the uncompressed nonwoven fabric layer or the porous collagen layer may be integrated. In particular, it is preferable to simultaneously press and compress those in which the nonwoven fabric layer and the sponge layer are integrated in the final step of film formation. When the collagen film is actually used at a surgical site or the like, the thickness of the film is reduced by pressurizing and compressing. This is because the handling is improved and the transplant operation or the like can be performed more smoothly.

In order to further improve the physical strength of the nonwoven fabric layer or the compressed nonwoven fabric layer obtained by the above method, the nonwoven fabric layer or the compressed nonwoven fabric layer is impregnated with a solubilized collagen aqueous solution. By performing drying by a suitable drying method such as drying, drying under air blowing, drying under reduced pressure, and drying under low temperature, it is possible to produce a medical collagen membrane in which the fiber gap of the nonwoven fabric layer is filled with collagen. That is, it is a medical collagen membrane in which both sides of a nonwoven fabric layer in which the fiber gap of a nonwoven fabric composed of crosslinked collagen fibers is filled with collagen are covered with a collagen coating. The physical strength of the medical collagen membrane obtained by this operation is much higher than that of the nonwoven fabric single layer, and the suture strength is also significantly improved. The step of impregnation and drying may be repeated one to several tens of times depending on the required physical strength. At this time, if the nonwoven fabric layer or the compressed nonwoven fabric layer is not subjected to a crosslinking treatment, the nonwoven fabric layer itself may be dissolved when the nonwoven fabric layer is impregnated with the solubilized collagen aqueous solution. After the cross-linking treatment, the nonwoven fabric layer is preferably impregnated with a solubilized collagen aqueous solution. In addition to the impregnating treatment, there are a method of casting or filling an aqueous solution of a solubilized collagen into a nonwoven layer contained in an appropriate container, and a method of directly applying the solubilized collagen solution to the nonwoven layer. By lyophilizing the medical collagen film in which both sides of the nonwoven fabric layer of the nonwoven fabric composed of crosslinked collagen fibers thus formed are filled with collagen, the both surfaces of which are covered with collagen coating. , Collar
A porous collagen layer having a porous gen coating portion is formed.

Alternatively, a film-like material may be obtained by sandwiching a nonwoven fabric layer with a collagen layer previously dried in a sponge form, or the porous collagen layer and the nonwoven fabric layer may be simultaneously compressed to form the nonwoven fabric layer. -Disposing the porous collagen layer of solubilized collagen by placing the multilayer obtained by embedding in the gen layer under normal pressure or reduced pressure in the presence of a dilute solubilized collagen aqueous solution or water, There is a method in which the nonwoven fabric layer is sufficiently blended with the nonwoven fabric layer and then dried by various drying methods. This method of using a porous collagen layer involves filling collagen into the interstices of the non-woven fabric layer, and requires a very small amount of a solvent component such as water with respect to the amount of collagen actually used. In addition, there is an advantage that the drying can be performed in a short time in a post-process, and contracture and deformation during the drying are very small. In the case of a normal impregnation step, the actual amount of collagen in the solubilized collagen aqueous solution for impregnation is limited to about several percent from the viewpoint of practical solution viscosity, and the remaining 90% or more is a solvent component such as moisture. In addition, it takes a long time to perform the impregnation and drying operations, and it is necessary to repeat the operation itself, which is a simple method but lacks rationality.

[0028]

EXAMPLES Examples of the present invention will be described below with reference to examples.

EXAMPLE 1 Distilled water for injection was added to a chicken-derived atelocollagen with gentle stirring with a magnetic stirrer to prepare aqueous collagen solutions of 3 wt% and 5 wt%. Next, 40 ml of a 5 wt% collagen aqueous solution was applied to the solution using a dispenser (manufactured by San-A-Tech: EFD900).
2.5b from needle tip of gauge size (pore diameter 200μm)
Continuous extrusion spinning was performed in a 99.5 vol% ethanol solution (manufactured by Wako Pure Chemical Industries, Ltd.) under a constant pressure of ar. During the continuous extrusion spinning, the tip of the needle was moved randomly on an ethanol coagulation bath to form an aggregate in which the continuous collagen yarn solidified by spinning settled in a network structure on the bottom surface of the coagulation bath. Next, this was left for 1 hour to sufficiently coagulate, and then washed twice by exchanging the coagulation solution with ethanol.

The aggregate of fibrous collagen thus obtained is directly used as a vacuum dry oven (manufactured by EYELA:
VOS-300VD type) Oil rotary vacuum pump (ULVAC: GCD135)
-XA type) at room temperature under reduced pressure (less than 1 Torr) for 4 hours to obtain a fibrous collagen nonwoven fabric. Next, this nonwoven fabric is put into a sterilized aluminum packaging material, and 100 kgf / cm ² is applied with a high pressure jack (15-ton press machine manufactured by Inuchi Seieido Co., Ltd.).
To obtain a compressed nonwoven fabric of 7 cm × 4.5 cm 2. Next, this compressed nonwoven fabric was immersed in a 5% solution of glutaraldehyde (a 25% solution of primary glutaraldehyde manufactured by Wako Pure Chemical Industries, Ltd. with distilled water for injection) for 4 hours to perform a crosslinking treatment. After the reaction is completed, wash thoroughly with distilled water for injection,
It was immersed in a distilled water bath for injection for 1 hour, and the water was changed three times during the immersion to remove excess glutaraldehyde. The crosslinked nonwoven fabric was dried at room temperature under reduced pressure (less than 1 Torr) for 4 hours and compressed.

Next, freeze-dried porous collagen was prepared. This is done by placing a 3 wt% aqueous solution of solubilized collagen into a mold and filling it to a thickness of about 17 mm.
(MEDICALFREEZER) at -20 ° C for about 12 hours. Next, the frozen solubilized collagen was transferred to a freeze dryer (manufactured by EYELA: FDU-830) while being kept in a container, and reduced under pressure (0.05 Torr) with an oil rotary vacuum pump (manufactured by ULVAC: GCD200-XA). ) For about 24 hours. The thickness of the porous collagen layer at the end of freeze-drying was about 15 mm. The obtained porous collagen layer and the cross-linked compressed non-woven fabric were overlapped and compressed at a pressure of 100 kgf / cm ² to obtain a multilayer collagen membrane having a size of about 7 cm x 4.5 cm in which the non-woven fabric layer was embedded in the porous collagen layer. Was.

Next, a very small amount of about 15 ml
Immersed in a 3 wt% aqueous solution of solubilized collagen in a vacuum dry oven (EYELA: VOS-300VD type), and decompressed the air in the multilayer collagen membrane to force the solubilized collagen aqueous solution. Impregnated. The multilayer collagen membrane in which the porous collagen layer thus obtained was dissolved was dried at a low temperature (4 ° C.) for 24 hours. The multilayer collagen membrane obtained by drying this is transferred to a container, a 3 wt% aqueous solution of solubilized collagen is poured from above, and the position is adjusted with sterile forceps or the like so that the multilayer collagen membrane almost comes to the intermediate layer. The mixture was frozen at -20 ° C. for about 12 hours in a freezer (manufactured by SANYO: MEDICALFREEZER). Keep the frozen collagen membrane in the container under reduced pressure (less than 0.05 Torr) for about 24 hours.
Lyophilization was performed for hours. A multilayer collagen membrane obtained by integrating the obtained compressed non-woven fabric layer and porous collagen layer with 400 kgf
/ Cm ² pressure, thickness about 1.5mm, size about 7cm ×
A 4.5 cm 3 multilayer collagen membrane having a three-layer structure was obtained. Next, the obtained membrane was subjected to thermal dehydration crosslinking treatment at 135 ° C. under reduced pressure (less than 1 Torr) for 12 hours using a vacuum dry oven and an oil rotary vacuum pump (type GCD135-XA, manufactured by ULVAC). In this way, a medical collagen membrane consisting of only collagen, in which only the fibers of the nonwoven fabric layer were subjected to the glutaraldehyde crosslinking treatment, was obtained.

[0032]

Example 2 As in Example 1, chicken atelocollagen was slowly stirred with a magnetic stirrer, and distilled water for injection was added to prepare collagen aqueous solutions of 3 wt% and 5 wt%. Next, 40 ml of a 5 wt% collagen solution was dispensed (manufactured by San-A-Tech: EFD9).
Using a 200 gauge (type 00), continuous extrusion spinning was performed from a needle tip of a 20 gauge size (pore diameter 600 μm) into a 99.5 vol% ethanol solution (■ Wako special grade) under a constant pressure condition of 2.0 bar. Thereafter, after obtaining a nonwoven fabric in the same manner as in Example 1, glutaraldehyde treatment was performed in the same manner for 4 hours, followed by compression again. A laminated structure was formed by using two pieces of the compressed nonwoven fabric thus obtained.
Next, in the same manner as in Example 1, solubilized collagen was impregnated into each nonwoven fabric layer using porous collagen, and dried at 4 ° C. for 24 hours to obtain a multilayer collagen film.

Next, this multilayer collagen membrane was immersed in a 5% solution of glutaraldehyde (Wako Pure Chemical Industries, Ltd., 25% solution of primary glutaraldehyde diluted with distilled water for injection) for 4 hours to carry out crosslinking treatment. . After completion of the reaction, the plate was sufficiently washed with distilled water for injection, and then immersed in a distilled water bath for injection for 2 hours. During the immersion, the water was changed five times to remove excess glutaraldehyde. After washing the multilayer collagen membrane,
After drying at 24 ° C. for 24 hours, the multilayered collagen membrane was transferred to a container, a 3 wt% aqueous solution of solubilized collagen was poured from above, and the position was adjusted with sterile tweezers or the like so that the multilayered collagen membrane almost came to the intermediate layer. Thereafter, the mixture was frozen at -20 ° C for about 12 hours in a freezer (manufactured by Sanyo: MEDICALFREEZER). Lyophilization was performed for about 24 hours in the same manner as described above, with the frozen collagen membrane still in the container. The obtained collagen membrane in which the compressed non-woven fabric layer and the porous collagen layer were integrated was compressed again at a pressure of 400 kgf / cm ² again to a thickness of about 1.
A collagen film having a thickness of 7 mm and a size of about 7 cm × 4.5 cm and having a four-layer structure was obtained. Next, the obtained membrane was subjected to a vacuum dry oven and an oil rotary vacuum pump (manufactured by ULVAC: GCD135-XA).
) At 135 ° C under reduced pressure (less than 1 Torr) for 12 hours. In this way, a medical collagen film consisting of only collagen, in which all layers of the nonwoven fabric layer were cross-linked with glutaraldehyde, was obtained.

Measurement of Suture Strength A measurement test was performed to determine the strength of the collagen membranes prepared in Examples 1 and 2 for sutures. The sample to be measured is the collagen membrane prepared in Examples 1 and 2. Further, as comparative examples, GORE-TEX pericardium (GORE-TEX: GORE-TEX EPTFE patch II (sheet for pericardium)), porcine isolated pericardium, and porcine isolated brain dura were used. The porcine isolated pericardium and excised dura were measured in a fresh state immediately after removing about 20 kg of a pig from a porcine under anesthesia by extirpation surgery, immersing it in physiological saline. The measuring method is as follows. First, the membranes of Examples 1 and 2 and the membrane of the comparative example were all cut out as plate-shaped sections of 1 cm × 2.5 cm, and a distance of 5 mm from one end in the long side direction, and a 4-0 prolene yarn (ETHICON, (Made by INC).
Next, after immersing this in physiological saline at 37 ° C. for 30 minutes,
Remove the sample immediately and use the Shimadzu Autograph S-500D.
Was used to measure the tensile strength. The measurement conditions were as follows: the end opposite to the side through which the 4-0 pro-lean thread passed was chucked and fixed to a distance of about 10 mm from the end, and the ring-shaped 4 at the other end was fixed.
A −0 pro-lean yarn was hooked on a measuring hook and subjected to a tensile measurement at a constant speed of 10 mm / min. At this time, the change in strength until the membrane was cut by the 4-0 prolean thread or the thread was separated from the membrane was measured, and the highest value among the recorded strengths was adopted as the suture strength of the membrane used for the measurement. Table 1 shows the measurement results. The unit is N.

[0035]

[Table 1]

As is clear from Table 1, the suture strength of the collagen membrane for medical use obtained in Examples 1 and 2 of the present invention is higher than that of the porcine pericardium of Comparative Example 2, and the commercially available product of Comparative Example 1 and Comparative Example 3 is slightly better than the porcine dura, indicating that it can be put to practical use.

Implantation test The collagen membrane obtained in Example 1 was used in rats (n = 8).
Was implanted subcutaneously in the back, and the tissue reaction was observed with the naked eye and an optical microscope to evaluate biocompatibility. For the implanted sample, the collagen membrane obtained in Example 1 was 5 mm × 15 mm.
It was cut into a size of mm and used. Comparative Example 4 was 3 wt%
Using the collagen aqueous solution of Example 1,
The same film as the gen layer was prepared, and the film processed into a film by performing a compression treatment without cross-linking was cut into the same size as the sample and used. Each of the membranes of Example 1 and Comparative Example 4 was sterilized by irradiating 25 kGy of γ-ray, and then used for an implanting test. Implantation involves first injecting anesthesia into the peritoneum of a rat (250-300 g),
Next, two incisions were made in aseptic manner, about 1.0 cm apart from each other across the spinal cord of the back of the rat and parallel to the spinal cord direction to expose the lower paraspinal muscle. Next, the fascia was incised, and the membrane of Example 1 or the membrane of Comparative Example 4 was inserted sideways from the incision, and each of the left and right incisions was placed so that the membrane did not hit the incision.
The membranes were implanted one by one.

The implantation site of the membrane of Example 1 or the membrane of Comparative Example 4 was switched left and right for each rat, and immediately before implantation, an appropriate amount of physiological saline was sprinkled on each membrane to make the membrane wet, and the flexibility was increased. The plants were buried in a raised state. The incision after implantation was closed with suture. After 1, 2, and 4 weeks after implantation, the sample was taken out under anesthesia, and anesthetic was further administered as it was and euthanized. When removing the membrane of Example 1 or the membrane of Comparative Example 4, the state of the membrane of Example 1, the membrane of Comparative Example 4, and the state of the surrounding tissue in both the implanted state immediately after the incision of the implanted site and the state after excision. Observed visually. In addition, the membrane of Example 1 and the membrane of Comparative Example 4 were excised together with the surrounding tissues, and then the inflammatory state and the state of the collagen membrane were observed with the naked eye and an optical microscope. In all rats, the wound at the incision was almost completely healed in about 2 to 3 days after implantation. Implanted Comparative Example 4 membrane was almost completely decomposed and absorbed after one week,
No inflammatory reaction was found in the surrounding tissue with the naked eye, and no fibrous scar was seen. Observation with an optical microscope showed a very small distribution of giant cells in the surrounding tissue of what appeared to be Comparative Example 4 membrane. Two or four weeks after implantation, no remaining collagen film could be confirmed by visual inspection or palpation, no inflammatory reaction was observed, and no fibrous scar was found. In addition, even by observation with an optical microscope, no aggregation of giant cells or the like was observed in the tissue surrounding the implant.

On the other hand, in the case of the membrane of Example 1, one week after implantation, the presence of the membrane could be clearly confirmed with the naked eye, and the shape could be recognized by palpation. No inflammatory response was noted with the naked eye, and no fibrous scars were found. No particularly remarkable inflammatory reaction or the like was observed by observation with an optical microscope. Two weeks after implantation, macroscopic and light microscopic findings were almost the same as one week after implantation, but slight evidence of decomposition and absorption was observed in the porous collagen layer. Four weeks after implantation, the sponge layer could be visually confirmed to remain, but decomposition and absorption had progressed. Although the non-woven fabric layer maintained its shape, it was still slightly decomposed and absorbed. Macroscopic observation showed no particular inflammatory response and no fibrous scar. Observation with an optical microscope showed a very slight distribution of giant cells in the tissue surrounding the implant. From the above results, the collagen membrane prepared in Example 1 had a remarkable inflammatory reaction even when implanted in the back of the rat,
No fibrous scars were found, demonstrating very good biocompatibility.

[0040]

Industrial Applicability The collagen film for medical use of the present invention has a laminated structure of a nonwoven fabric layer and a coating layer, using collagen, which is a biological material, as a main raw material. Then, the nonwoven fabric layer has a sutable film strength of the medical collagen film, and the coating layer is involved in the improvement of biocompatibility and tissue regeneration promoting performance. The present invention has solved all the deficiencies of sufficient biocompatibility and tissue regeneration promoting performance.

Claims (8)

[Claims] 1. A medical collagen membrane wherein at least one surface of a non-woven fabric layer comprising cross-linked collagen fibers is covered with a collagen coating. 2. The collagen membrane for medical use according to claim 1, wherein the diameter of the cross section of the collagen fiber constituting the nonwoven fabric is 10 to 1000 μm. 3. The medical collagen membrane according to claim 1, wherein the collagen coating comprises a porous collagen. 4. A nonwoven fabric layer comprising a crosslinked collagen fiber and said fiber gap filled with collagen.
A medical collagen membrane according to any one of the above. 5. The method according to claim 1, wherein the collagen is an enzyme-solubilized collagen,
The medical collagen membrane according to any one of claims 1 to 4, wherein the collagen membrane is at least one selected from acid-solubilized collagen, alkali-solubilized collagen, and neutral solubilized collagen. 6. The medical collagen membrane according to claim 1, wherein the porous collagen coating is obtained by freeze-drying. 7. The medical collagen membrane according to claim 1, wherein the collagen coating is crosslinked. 8. The medical collagen membrane according to claim 1, wherein the collagen filled in the nonwoven fabric layer is crosslinked.