JPH0669486B2 - Method for producing collagen membrane having biological function - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to a method for producing a collagen membrane, specifically, to reconstitute soluble collagen into a fibrous form, reduce and stabilize it, and then dry it to produce a membrane. A method for producing a collagen film, and
The present invention relates to a method for producing a composite collagen membrane, which is produced by mixing a connective tissue component other than collagen into the membrane, and the membrane includes periodontal tissue (alveolar bone, cementum, periodontal ligament) destroyed by periodontal disease and the like. It can be used as various bio-repair materials.

(Conventional technology) Fibrous protein collagen is a biological protein widely distributed in connective tissues of mammals, birds, etc., bones, teeth, ligaments, tendons, dermis, fascia, etc. Recently, various medical and biological applications have been investigated. Has been done. For example, reconstituted collagen is not only an excellent support for cell culture, but is also effective for giving swelling to the depressed skin surface (so-called wrinkle) by injecting subcutaneously into the human body. Thus, various soluble collagens are widely used in hair, cosmetic, household, animal, and industrial products. A collagen film, such as metaskin, is used in medicine as a therapeutic agent for coating burns and skin defect wounds.

However, the collagen membranes currently used are rather rigid, hard to adapt to human tissues, and lacking in softness. For this reason, it is necessary to soften with physiological saline during use, and depending on the type of membrane, it is necessary to distinguish the front and back of the membrane when applied to the wound, and the exudate accumulates between the wound surface and the membrane,
The adhesion to the wound surface may be poor. Due to these drawbacks, it has been found that when collagen is applied to a wound, it promotes epidermal formation due to its self-absorbing property and protective effect, and promotes treatment of the wound surface, but it is not often used. Absent. Especially, it is not used for treatments that require fine treatment such as treatment of periodontal diseases in the dental field. Therefore, various studies have been added to improve these drawbacks.

(Problems to be Solved by the Present Invention) The present invention, except for the drawbacks of conventional collagen membranes, is flexible and absorbs exudative tissue fluid and has good adhesion to the wound surface, and when used, its effect of promoting epidermal formation can always be expected. The present invention establishes a method for producing a collagen film that can be used for fine treatment and a method for regenerating alveolar bone, cementum, and periodontal ligament destroyed by periodontal disease using the film.

(Means for solving the problem) Collagen molecules do not exhibit high elasticity like keratin, but as a structural feature, three polypeptide chains are helically aligned around the central axis, which is characteristic of collagen. Creating the structure (collagen helix). It is known that collagen fibers have a regular arrangement of collagen molecules and form a cross-link mainly having a Schiff base type through lysine and hydroxylysine to stabilize the structure. It is also known that soluble collagen is an existing form as a monomer of a collagen molecule, and collagen fiber is an existing form in which molecules have an extremely ordered arrangement and express the original performance of collagen. Therefore, in the conventional collagen membrane, the soluble collagen solution is dehydrated and dried as it is by a conventional method, so that the obtained membrane has collagen randomly coordinated in an arbitrary direction, and the original function of collagen cannot be exhibited. That is clear. Therefore, it has rigidity as a physical property and lacks flexibility. Therefore, various kinds of methods for giving the membrane the original physical properties of collagen and also giving elasticity and flexibility as physical properties were investigated.

As a result, the soluble collagen solution was reconstituted into a fibrous form in a flat container and gelled, and then the aldol condensate and the Schiff-type crosslinks present in collagen were reduced and stabilized by a mild reducing agent. , Collagen rapidly increases in strength to become a reinforced gel, and further, various growth differentiation factors including components of connective tissue other than collagen, such as bone morphogenetic protein (BMP), and hyaluronic acid, chondroitin sulfate, By adding intercellular matrix components such as the adhesive proteins fibronectin and osteonectin to the collagen membrane, it is possible to further impart a biological function. Membranes obtained by gradually dehydrating and drying these with ethanol etc. by a conventional method. Knew that it would be a soft and soft cloth-like structure instead of a film ( This was to make the film referred to as a cross-linked collagen sheet).

That is, the present application aims at a method for producing a crosslinked collagen sheet which is basically different from the conventional method in that it has a biological function and its use. The thickness of the cross-linked collagen sheet can be arbitrarily adjusted by the thickness of the gel, and its strength and flexibility can be adjusted by the type of reducing agent and connective tissue component used and its processing conditions. Soluble collagen derived from cattle, pigs, and other animals and collagen excluding the telopeptide at the molecular end or binding components to this collagen such as bone morphogenetic protein (BMP), hyaluronic acid, chondroitin sulfate, adhesive protein fibronectin, osteo Dissolve substances such as Nectin in neutralization buffer
Adjust pH to neutral. The collagen concentration is not particularly limited, but a high concentration solution causes a problem in handling, so 1%
The concentration is adjusted to the following, preferably about 0.2%.
In addition, it is preferable to add various binding compositions to collagen because it improves the strength and flexibility of the obtained membrane. The ratio of addition to the collagen can be arbitrarily changed according to the properties of the target film, and is not particularly limited,
A relatively small amount may be added, and generally 10% by weight or less based on the weight of collagen is sufficient. The solution is sterilized by filtration and then poured into a flat plate container placed horizontally to cause gelation. About 37
Allow to gel overnight at ℃. The gel obtained is kept in a neutral buffer and treated with a reducing agent, for example a weak reducing agent such as a borohydride compound, preferably sodium borohydride. In order to increase the strength and flexibility of the crosslinked collagen sheet, it is possible to stabilize physiological crosslinking with a reducing agent and artificially introduce crosslinking with a crosslinking agent such as hexamethylene diisocyanate. The type of the cross-linking agent and its treatment conditions are selected according to the properties of the cross-linked collagen sheet of interest, but when sodium borohydride is generally used as the reducing agent, the amount thereof is about 1/50 to 1 of the collagen used. / 10 (weight), preferably about 1/30 amount, at a temperature of 50 ° C. or lower, preferably about room temperature for 1 to several hours, when hexamethylene diisocyanate is used, 1/20 to 1/10 amount of collagen It is treated at room temperature for 1 to several hours. The gel treated with a reducing agent is washed with water to completely remove the reducing agent, and then dehydrated with ethanol is repeated by a conventional method, and finally dried under reduced pressure at a low temperature to obtain a crosslinked collagen sheet.

When the crosslinked collagen sheet thus obtained is attached to a tissue, it is finally absorbed by the tissue, and since it is a soft cloth and has a high strength, it can be attached to any tissue and adheres thereto. Further, collagen has a low immunogenicity, and becomes even lower when the telopeptide at the molecular end is removed, and has a low affinity as a foreign substance to cells and an excellent affinity. Therefore,
When this crosslinked collagen sheet is used as a regenerating material for periodontal tissue on the tooth surface during periodontal disease gingival flap exfoliation surgery, regeneration of periodontal tissue after surgery is remarkably good and treatment of periodontal disease is promoted. That is, the connective tissue component collagen sheet compensates for the shortcomings of the non-absorbable Millipore filter which has been conventionally used for partly in periodontal treatment, and enables use as a new material to replace it. Fig. 1 is a diagram of periodontal disease in which gingival epithelium is not normally attached, Fig. 2 is a diagram in which a crosslinked collagen sheet is attached to teeth during gingival flap detachment surgery, and Fig. 3 is gingival and alveolar bone.・ The figure shows that the cementum and periodontal ligament were regenerated and the gums were correctly attached to the teeth.
Since the cross-linked collagen sheet was adhered to the tooth surface, the epithelium did not show unnecessary proliferation toward the root of the tooth, remained in the original position of the epithelium, under which alveolar bone and cementum were regenerated, and the periodontal ligament was correctly formed. Wraps around the roots. Therefore, the invasion of the gingival epithelium in the direction of the tooth root as shown in FIG. 1 does not occur, which is extremely effective in treating periodontal diseases.

(Function) The crosslinked collagen sheet is produced by reconstituting soluble collagen into fibers, treating it with a reducing agent, and further artificially introducing connective tissue components as necessary. For this reason, unlike the conventional one in which collagen molecules are dried in a disordered state, the collagen membrane according to the present application retains its biological function, is rigid, and has a flexible and strong cloth-like shape. Suitable for Hereinafter, the present invention will be described with reference to examples.

Example 1. 0.3% acid soluble collagen 0.1 from bovine skin tissue
After sterilizing 7 volumes of M acetic acid solution (pH 3) by filtration, 1 volume of 0.5 M HEPES buffer and 2 volumes of 1 M NaOH were mixed to homogenize, and after confirming that the pH was neutral, 20 × 10 cm It was poured evenly on a flat plate having a bottom area, and the flat plate was left overnight in a constant temperature bath at 37 ± 1 ° C. to obtain a gel. Apply this gel to pH 7 phosphate buffer 400
It was kept at ml, 140 mg of NaBH ₄ was added, and it was kept at room temperature for 1 hour. After removing the aqueous layer, the product is washed with water to remove the reducing agent and dehydrated with ethyl alcohol by a conventional method. After dehydration with ethyl alcohol, the product is dried under reduced pressure at room temperature to a thickness of about 20.
A μ collagen film was obtained. It was less rigid and more flexible than a commercially available product (Metaskin).

Example 2. The collagen film obtained in Example 1 was attached and adhered to the tooth surface at the time of periodontal disease gingival flap exfoliation surgery as shown in FIG. When a membrane is used, the apical movement of the gingival epithelium is blocked and young cells are differentiated and grown from the periodontal ligament and bone to regenerate alveolar bone, cementum and periodontal ligament. On the other hand, when the membrane was not used, the epithelium grew downward as shown in FIG. 1, and the regeneration of alveolar bone, cementum and periodontal ligament occurred only slightly.

(Effect) Since the crosslinked collagen sheet obtained by the present invention is strong, flexible and cloth-like, it can be applied to any fine operation and is excellent as artificial skin.

In addition to the artificial periodontal ligament and the artificial skin, the crosslinked collagen sheet obtained by the present invention is sufficiently effective for the following medical applications. From human dura in the past,
There is a film commercially available for clinical use by peeling the collagen film as it is, and it has been widely applied. However, this material was obtained from post-mortem material in humans and is now almost unusable due to AIDS problems. As an artificial membrane that replaces this, the one described in the present application can be used.
Further, applicable examples will be illustrated below.

Repair of periodontal tissue destroyed by periodontal disease Protection of gingival supply site during gingival transplantation Replacement of dura in neurosurgery Closure of cerebrospinal fistula Replacement of orbital bed (hammock formation) Wall formation of maxillary sinus Mouth vestibular repair Replacement of the pericardium External abdominal defect of the diaphragm, closure of congenital hiatus Fascia strengthening or replacement of abdominal wall hernia Replacement and strengthening of the tendon proximal, encapsulation to prevent adhesions Plastic expansion of the bladder Coverage during Achilles tendon tear surgery Package strengthening Other

[Brief description of drawings]

Fig. 1 shows that periodontal disease in which deep periodontal pockets are formed, alveolar bone is absorbed, cementum and periodontal ligament are greatly destroyed, and gingiva is not properly attached to the tooth surface. It is sectional drawing of a tooth. FIG. 2 is a cross-sectional view of a tooth in which the cross-linked collagen sheet of the present application is stuck and adhered from the tooth to the surface of the bone at the time of gingival flap peeling surgery for a tooth with periodontal disease. FIG. 3 is a sectional view of a completely cured tooth. In the figure 1. Alveolar bone 2. Cementum 3. Periodontal ligament 4. Gingiva 5. Periodontal pocket 6. Crosslinked collagen sheet 7. Regeneration of alveolar bone 8. Regeneration of cementum 9. Regeneration of periodontal ligament

Claims (2)

[Claims] 1. A method for producing a collagen membrane, which comprises reconstituting soluble collagen into a fibrous form and treating it with a borohydride compound. 2. A composite collagen produced by mixing at least one selected from bone morphogenetic protein (BMP), hyaluronic acid, chondroitin sulfate, adhesive protein fibronectin and osteonectin during the production of the collagen membrane according to claim 1. Membrane manufacturing method.