JPH0818998B2 - Collagen therapeutic material for dental and oral surgery - Google Patents

Description

The present invention relates to treatment of periodontal disease in the fields of dentistry and oral surgery, prevention of growth of gingival epithelium in the apical direction of the root, that is, prevention of down-growth and treatment of extraction tooth sockets. The present invention relates to a therapeutic collagen composition.

(Prior Art and Problems to be Solved) The ultimate purpose of periodontal treatment is to regenerate periodontal tissue destroyed by periodontitis or the like. When only normal surgical treatment is performed, the healing form is that the cells that produce cementum and periodontal ligament do not specifically reach the root surface before the epithelium,
First, the epithelium embeds along the connective tissue on the root surface, resulting in the formation of long junctional epithelia. This fibrous attachment without cementum or periodontal ligament is fragile,
It does not completely restore the occlusal function of the teeth. Furthermore, the direct attachment between the root epithelium and the root causes root absorption.

Therefore, based on the idea of isolating the tooth root from other tissues and selectively collecting the cementum and cells that generate periodontal ligament on the surface of the tooth root, attempts to insert various membranes between the tooth root and the gingiva have been made. It has been done. For example, Millipoer Filter
When Gore-Tex membrane is used, cementum and periodontal ligament are certainly regenerated around the root, but the membrane itself is non-absorbable in vivo, so it is after membrane transplantation to regenerate gingival connective tissue and epithelium. At that time, a surgical operation for removing the membrane had to be performed again, and the operation was complicated, which was not very practical. In addition, there is a report using an atelocollagen sheet as an example of a collagen film (Kuichi Kamoi et al., Tokyo Dental Association Magazine: Vol. 36, No. 4, April 1988). Since collagen is biodegradable, re-operation for extraction is not necessary, and an ideal healing form was shown in which cementum and periodontal ligament exist around the tooth root and connective tissue adheres to it. . However, in some cases, before the cementum and periodontal ligament are regenerated, they may be decomposed in vivo, or even if they are not decomposed, the morphology of the film may not be maintained due to expansion by body fluid. In addition, due to the lack of strength of the membrane itself, it was difficult to handle the insertion, because it required a skilled skill and careful attention.

By the way, collagen is a major protein that constitutes connective tissue of animals, and its structure has almost the same structure regardless of animal species. Therefore, even collagen derived from xenogeneic animals is characterized by extremely low antigenicity and excellent tissue compatibility unlike other proteins. Generally, collagen that constitutes a tissue has crosslinks between its molecules and maintains the form as a tissue. Atelocollagen cuts this intermolecular bridge by treating with an enzyme,
Furthermore, the telopeptide which is an antigen expression site is removed.

As shown above, collagen, particularly atelocollagen, has extremely excellent tissue compatibility, but as it is, it has a drawback that it is rapidly decomposed and absorbed in vivo and is decomposed and absorbed before the therapeutic purpose is sufficiently fulfilled. . Crosslinking is introduced by various methods in order to control the decomposition and absorption rate in the living body. However, even if the biodegradability and absorption can be controlled by introducing crosslinks, the strength of the composition could not be sufficiently improved to a desired level.

The present invention provides a therapeutic collagen composition in the dental and oral surgery fields, which has a sponge-like and sheet-like structure composed of collagen, has sufficient strength, and has controlled degradation and absorption in vivo. To aim.

(Means for Solving the Problems) The present inventor has conducted various studies on the strength and bioabsorption rate of collagen membranes, and as a result, soluble collagen was blended with fibrillar collagen at an appropriate ratio and crosslinked with a crosslinking agent. The inventors have found that the strength and the rate of bioabsorption are extremely improved, and have completed the present invention.

That is, the present invention relates to soluble collagen 95 to 40% by weight.
And a collagen composition comprising 5 to 60% by weight of fibrous collagen, which is a crosslinked collagen composition for dental and oral surgery.

Soluble collagen used in the present invention, collagen extracted with acid from mammals such as cows, rats, mice, etc., further birds, fish, and the like, or dissolved and extracted using enzymes from the connective tissue of these animals. Atelocollagen or collagen extracted by alkali treatment,
Furthermore, such as collagen derivatives chemically modified,
It can be dissolved in water to form a uniform aqueous solution. In particular, atelocollagen obtained by solubilizing bovine dermis with pepsin is preferable.

Moreover, as the fibrous collagen used in the present invention, tendon-derived collagen isolated from tendon, cross-linked collagen obtained from the dermis layer of animal skin, or the like can be used. In particular, it is desirable to use tendon-derived collagen.
The collagen derived from tendon has long fibers, and when used in combination with soluble collagen, the mechanical properties of the molded product can be greatly improved.

Furthermore, the tendon has less proteins other than collagen as compared with other organs and is easy to purify. Purification of tendon-derived collagen can be performed by dissolving and separating fat etc. using an organic solvent, then using a protein denaturing agent to make collagen difficult to denature, and washing it under conditions where other proteins denature and elute. I found it. This has made it possible to safely apply fibrillar collagen to ecology.

As described above, the collagen composition composed of soluble collagen and fibrillar collagen can be used by mixing them at any ratio depending on the desired strength, mechanical properties such as flexibility, and biodegradation / absorption rate. , Soluble collagen 95-40% by weight and fibrous collagen 5-60% by weight
Is preferable, and particularly preferable mixing ratio is 90 to 70% by weight of soluble collagen and 10 to 30% by weight of fibrillar collagen.

This mixing operation is performed by adding an acidic suspension having a concentration of 2% or less of fibrous collagen to an acidic aqueous solution having a concentration of 5% or less of soluble collagen and dispersing it. At this time, the concentration of total collagen is preferably 3% or less. This operation is performed by stirring with a homogenizer while paying attention to the temperature rise so that the solution temperature does not rise above 20 ° C. This uniform acidic dispersion is, as it is, or neutralized and freeze-dried or air-dried to form a sponge or sheet. After that, press to obtain a thickness of 500μ when swollen.
It should be less than m.

If the uncrosslinked collagen membrane is directly applied to a living body, it will swell due to body fluid and the like, and it will be difficult to handle, and it will be decomposed and absorbed within about one week after transplantation. Therefore, the collagen sponge is crosslinked. As the cross-linking agent, conventionally well-known ones, for example, aldehyde compounds such as formaldehyde and glutaraldehyde, isocyanate compounds such as hexamethylene diisocyanate, and polyepoxy compounds can be used. Particularly preferred is hexamethylene diisocyanate, which has no irritation such as cytotoxicity. Regarding the degree of crosslinking, it is desirable that the shape of the membrane is maintained for 3 weeks or more after transplantation.

Example (1) Preparation of atelocollagen Fresh calf skins were thoroughly washed with purified water to remove adhered dirt. After disinfecting with 70% ethyl alcohol, the hair was removed and the surface of the skin was scraped off with a razor so as not to leave the root of the hair, and the back surface was also similarly processed to remove the fat layer and the like to obtain a dermis layer. Shred this, wash with 5% NaCl water, and then dermis 10
Distilled water of 4 was added to 0 g, pH was adjusted to 3.0 with 1N HCl, 0.2 g of pepsin was added, and the mixture was kept at 20 ° C. for 5 days while performing intermittent stirring.

The pH of the soluble collagen aqueous solution obtained by this treatment was adjusted to 10 in order to inactivate pepsin (NaO
H) Hold at 4 ° C for 24 hours.

Then, the isoelectric focusing method and the filtration were repeated to purify the collagen to obtain aseptic and pyrogen-free atelocollagen.

(2) Preparation of Tendon-Derived Collagen The sheath of fresh bovine Achilles tendon was removed with scissors. A Y-shaped tendon without a sheath was sliced with a meat slicer to a thickness of 1 to 2 mm. Treated with Micro-Cut 2-3 times for uniform and individual fiber separation. This operation reduced the fiber length to 0.2 mm or less. The collagen fibers of the micronized tendon were washed twice with a fatty solvent such as ethyl alcohol-ethyl ether to remove a fat component, and then collected by centrifugation. In order to remove impurities such as other proteins,
It was washed at 4 ° C. with a double amount of 1 M calcium chloride solution.
By this operation, for example, the content of albumin in collagen was 1 ppm or less.

Thereafter, washing with distilled water and collection were further repeated to obtain biocompatible fibrous collagen derived from tendon.

(3) Production of collagen sponge Using the atelocollagen prepared in (1) above, a 2% by weight acidic aqueous solution (pH 3; HCl) was prepared. Further, using the tendon-derived collagen prepared in (2) above, an acidic dispersion liquid (pH 3; HCl) having a collagen concentration of 2% by weight was prepared.
The weight ratio of the atelocollagen aqueous solution and the tendon-derived collagen dispersion is (a) 100: 0, (b) 90:10, (c) 60: 4.
0, (d) 40:60 were mixed. Then, the mixture was neutralized with NaOH while stirring with a homogenizer to obtain a dispersion having a pH of 7-8. Dispense this dispersion into 1 mm thick freeze-drying tray,
Lyophilization was performed. The sponge obtained was compressed to a thickness of 0.2 mm. Then, a crosslinking reaction was carried out by immersing the solution in a solution prepared by dissolving hexamethylene diisocyanate in a molar ratio of 5 times with respect to ε-NH _{2 of} collagen in methyl alcohol so as to be 0.05% and shaking. Thereafter, washing with methyl alcohol and washing with water were carried out, followed by drying to obtain a collagen sponge sheet having the composition (a)-(d). The collagen sponge sheet thus obtained had a cross-linking rate of ε-NH ₂ of about 55 to 60%.

(4) Comparison of Physical Properties of Collagen Sponge Sheet The tensile strength and flexibility of the collagen sponge sheets having the respective compositions (a) to (d) prepared in (3) above were measured. Tensile strength of each sponge sheet
It was cut into a size of 1 cm × 7 cm and measured by a tensile tester. Flexibility is evaluated by appearance and texture,
The collagen sponge sheet of (a) was set to 100, and this was used as a reference. The results are shown in Table 1.

(5) Bioavailability Male Wister rats (200-250g, 7 weeks old) were shaved on the back under anesthesia, and after disinfection with isodine, they were placed symmetrically with respect to the midline.
Subcutaneous pockets were created by making incisions of about 1.5 cm square at 4 locations each. Each of the collagen sponge sheets (composition) of (a) to (d) cut into 1 cm squares was placed and sutured.
Care was taken in this case not to place the skin incision line on the implant. After 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks and 7 weeks, the animals were dissected, and a sample of each subcutaneous tissue was taken out, fixed in formalin and embedded in paraffin to prepare a tissue section for histological evaluation. For comparison, the non-crosslinking product (e) of (b) was also evaluated. The results are shown in Table 2.

(Effect of the invention) Since the collagen therapeutic material of the present invention is a mixture of soluble collagen and fibrillar collagen in a specific ratio and is cross-linked, the collagen sheet or sponge having improved strength and bioabsorption rate. As a collagen therapeutic material for dental and oral surgery, for example, promotion of wound healing in periodontal treatment, prevention of downward growth of gingival epithelium or downgrowth, prevention of leakage of filler after filling of extraction tooth socket, etc. Extremely useful.

Claims (3)

[Claims] 1. A collagen or dental surgery collagen therapeutic material obtained by cross-linking a collagen composition comprising 95-40% by weight of soluble collagen and 5-60% by weight of fibrillar collagen. 2. The collagen treatment material for dental and oral surgery according to claim 1, wherein the soluble collagen is atelocollagen and the fibrous collagen is collagen derived from tendon. 3. The collagen or dental surgery material for dental and oral surgery according to claim 1, wherein the crosslinking is carried out with a crosslinking agent hexamethylene diisocyanate.