JP3048289B2 - Collagen-calcium phosphate composite material and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a collagen-calcium phosphate composite used as a tissue regeneration membrane (GTR membrane) in periodontal tissue, a hemostatic agent, a bone filling material, a cartilage filling material, a three-dimensional culture substrate for hard tissue cells, and the like. Materials and their uses.

[0002]

2. Description of the Related Art Conventionally, a material obtained by crosslinking collagen with a polyphenol-based crosslinking agent (for example, a collagen film or the like) has been used as a GTR membrane or a bone filling material. These materials are excellent in biocompatibility and flexibility because collagen is derived from living organisms. However, this material failed to promote tissue and bone repair.

Conventionally, collagen membranes, cotton-like materials or fibrin glue have been used as a hemostatic agent. However, particularly in the case of hard tissue, its hemostatic effect and subsequent hard tissue repair have been insufficient. On the other hand, α-tricalcium phosphate (hereinafter, referred to as “α-tricalcium phosphate”)
α-TCP), tetracalcium phosphate (hereinafter Te)
Calcium phosphate compounds having a chemical activity such as octacalcium phosphate (hereinafter referred to as OCP),
For example, in vivo or in the oral cavity, it is gradually converted into hydroxyapatite (hereinafter, referred to as HAp) or carbonate apatite (hereinafter, referred to as CO ₃ -Ap), which is a main component of the living hard tissue, and is integrated with the living hard tissue. It can be transformed.
However, a calcium phosphate compound having a chemical activity alone has poor flexibility and is difficult to mold, and it is difficult to appropriately supplement an affected part and has no hemostatic effect.

[0004] Therefore, if a calcium phosphate compound having the above-mentioned chemical activity is chemically bonded to a material using collagen as a main raw material, the collagen material has flexibility and moldability and can promote the repair of tissues and bones. Can be obtained. By the way, as the bio-adhesive, there was a fibrin glue, a gelatin-resorcinol-formaldehyde adhesive (GRF) and the like. However, fibrin glue has high biocompatibility but low adhesion. On the other hand, the GRF adhesive has strong adhesiveness but low biocompatibility, and may cause inflammation of an affected part and cell death.

[0005] The above-mentioned material obtained by chemically binding a calcium phosphate compound having a chemical activity to a material using collagen as a main raw material is also suitable for use as a hemostatic agent and a biological adhesive.

[0006]

SUMMARY OF THE INVENTION Accordingly, the present invention is intended to provide a composite material containing collagen capable of chemically binding calcium phosphate to a material containing collagen. An object of the present invention is to provide a composite material having both of the above and a use thereof.

[0007]

Means for Solving the Problems In order to solve the above problems, a collagen-calcium phosphate composite material according to the present invention is a composite material containing collagen, wherein a calcium phosphate compound having a chemical activity and a phosphate ion source are compounded. It is characterized by becoming. The collagen used in the present invention is not particularly limited, but it is preferable to use atelocollagen. Atelocollagen is collagen from which telopeptides at the molecular terminals have been partially or entirely removed by enzymatic treatment, and has no harm to living organisms. Collagen may be used as a solution or in a powder state.

[0008] The collagen concentration of the collagen solution is 0.
A range of 2 to 2.0% is preferred. If it exceeds this range, the viscosity is too high to provide as a solution, and if it is below this range, the effect of collagen is not exhibited because it is too thin. When collagen is used in a powder state, the average particle size is 32.
μm or less, preferably 10 μm or less. When the average particle diameter is large, it is difficult to dissolve the compound in the reaction solution, and it is impossible to uniformly disperse collagen.

In the present invention, a phosphate ion source is blended. It is usually added in the form of an aqueous solution. The phosphate ion source assists the crystal growth of a calcium phosphate compound having chemical activity at the time of complexation, and causes OCP, Ca-deficient HA
p, facilitating production of a CO ₃ -Ap. Sources of phosphate ions include potassium monophosphate, sodium phosphate dibasic, and the like. The molar concentration of phosphate ions in the aqueous solution is preferably from 2/1 to 1 / 30M. If the concentration is lower than this range, the crystal growth of calcium phosphate is delayed as described above, and if the concentration is higher, potassium monophosphate, dibasic sodium phosphate and the like are precipitated without dissolving. Further, a buffer solution or a culture solution (for example, a MEM medium solution, a BGJ-b medium solution, etc.) containing phosphate ions may be added.

As the calcium phosphate compound having a chemical activity used in the present invention, α-tricalcium phosphate (hereinafter referred to as α-TCP), tetracalcium phosphate (hereinafter referred to as TeCP), octacalcium phosphate ( Hereinafter, referred to as OCP). The calcium phosphate compounds having the above chemical activities may be used alone or in combination of two or more. These may be used in a powder state. The amount used is such that the weight ratio in the dried composite material is collagen: calcium phosphate compound = 99.9: 0.1 to
0.7: 99.3 is preferred. If the amount of the calcium phosphate compound exceeds this, good complexation with collagen is not performed.

In the present invention, a polyphenol-based crosslinking agent may be further added. Collagen is crosslinked by a crosslinking agent. As the cross-linking agent, a polyphenol-based cross-linking agent is desirable, and particularly, tannic acid, lignin and the like are preferable. Tannic acid and lignin are bio-related substances,
This is because it has no harm to living organisms. The amount of the crosslinking agent used is
0.05-5% is preferable with respect to collagen. Above this range, there is a possibility that the gel is partially cross-linked and conversely inhibits the gelation of collagen.

In the present invention, if necessary, ascorbic acid may be added. Ascorbic acid has a vitamin C effect in vivo, activates collagen synthesis and promotes cell growth. In addition, antibiotics such as tetracycline, anticancer agents such as cisplatin, b-FGF, TGF-β superfamily and their DNAs, cell growth factors and bioactive factors may be added to the composite material of the present invention.

The composite material of the present invention is obtained by drying a composite obtained by reacting the above components. It may be air-dried or freeze-dried using a solvent harmless to the living body such as ethanol. Further, a biological adhesive according to the present invention is obtained by adding gelatin and resorcinol to the composite material according to claim 1.

By adding gelatin, the strength after hardening can be increased. In addition, the carboxyl group of gelatin is attached to apatite, and biopolymer-complexed carbonate apatite (hereinafter, simply referred to as CO ₃ -Ap) is generated. This CO ₃ -Ap can completely replace living tissue. As the gelatin used in the present invention, Pharmacopoeia gelatin, Gelatin 21 (manufactured by Nitta Gelatin Co., Ltd.)
And the like, and pyrogen-free gelatin is particularly preferred. This is because pyrogen-free gelatin is less harmful to living organisms. Here, pyrogen-free gelatin is gelatin from which exothermic substances such as bacterial endotoxins (high-molecular-weight lipopolysaccharide) have been removed. Pyrogen-free gelatin is obtained, for example, by subjecting ossein or animal hide, which is a raw material of gelatin, to alkali treatment and washing with pyrogen-free water, and then extracting gelatin (see US Pat. No. 4,374,063). It can be obtained by, for example, a method of removing pyrogen by filtration through an outer filtration membrane (see JP-A-56-68607).

The above-mentioned gelatin may be added as it is in a powder state or may be used as a solution. The amount added is not particularly limited. In this invention, resorcinol acts as an adhesive component. Resorcinol is less harmful to living organisms. The addition amount is preferably 0.1 to 20% (weight ratio). If the concentration is higher than this, harm to the living body cannot be neglected, and the elasticity of the adhesive after cross-linking decreases, operability deteriorates, and the adhesive easily peels off from the application site.

[0016]

The collagen-calcium phosphate composite material according to the present invention is excellent in biocompatibility and flexibility because it is mainly composed of collagen constituting a living hard tissue. Furthermore, since calcium phosphate having chemical activity is added to this, it is replaced with living tissue over time. This is because calcium phosphate having chemical activity synthesizes CO ₃ -Ap and OCP, which are the main components of living hard tissue, with the hydrolysis reaction.

Further, since collagen having gelation energy is hydrolyzed with calcium phosphate having crystallization conversion energy to form a complex, both effects can be obtained well. Calcium phosphate having chemical activity undergoes a hydrolysis reaction in an aqueous solution containing phosphate ions, and is crystallized by a phase transition reaction over time on a crystal interface. In this crystal conversion,
In the neutral pH range, metastable OCP is generated and grows. The OCP couples with a sol → gel phase transition reaction of collagen having a carboxyl group to form an interlayer composite.

The biological adhesive according to the present invention has high biocompatibility and high adhesiveness because gelatin and resorcinol are added to the composite material of the present invention.

[0019]

Embodiments of the present invention will be described below, but the present invention is not limited to the following embodiments. -Example 1-Phosphate buffer (pH
To 7), UV-sterilized OCP was added at a ratio of OCP / phosphate buffer = 0.25 g / 10 ml, and the mixture was stirred for 1 hour. Next, a 0.3% aqueous solution (pH 3) of UV-sterilized collagen-IP (Cell Matrix-IP: manufactured by Nitta Gelatin Co., Ltd.) was added to a phosphate buffer: collagen solution = 1: 1.
And stirred for 5 minutes. The obtained composite was dispensed into a multiplate, and was placed in a CO ₂ incubator (37).
° C., 1 day CO ₂ = 5%) within 3 days, allowed to stand for 1 week.
These were subjected to 10% formalin treatment and 100% ethanol treatment and air-dried to obtain a composite material for the purpose of analyzing crystal conversion and crystal growth.

Example 2 The procedure of Example 1 was repeated except that a phosphate buffer containing 0.2% (W / V) of tannic acid was used. Example 3 Instead of OCP, α-TCP sterilized by dry heat at 150 ° C.
Was added in the same manner as in Example 1 except that α-TCP / phosphate buffer = 1 g / 10 ml was added.

Example 4 α-TCP sterilized by dry heat at 150 ° C. instead of OCP
Was added in the same manner as in Example 2 except that α-TCP / phosphate buffer = 1 g / 10 ml was added. Comparative Example 1 The procedure of Example 3 was repeated, except that instead of α-TCP, HAp sterilized by dry heat at 150 ° C. was used.

Comparative Example 2 The procedure of Example 4 was repeated, except that instead of α-TCP, HAp sterilized by dry heat at 150 ° C. was used. For Examples 1 to 4 and Comparative Examples 1 and 2, observation of gelation, freeze-drying and measurement of the following physical properties were performed. The results are shown in Table 1 and FIGS. [Measurement method] (1) pH measurement The pH of the gel composite collected at predetermined time intervals was determined by measuring the pH
It was measured using a meter. (2) X-Ray Diffraction The obtained composite material was identified using a powder X-ray diffractometer (MXP ³ , manufactured by Mac Science). (3) Scanning electron microscope The obtained composite material was scanned with a scanning electron microscope (CS-2100).
The surface was observed with a type A (manufactured by Hitachi, Ltd.).

[0023]

[Table 1]

1. Gelation As shown in Table 1, in the system to which tannic acid was not added, good gelation was exhibited at the initial stage, the hardness increased over time, and crystals were formed. On the other hand, in the system to which tannic acid was added, a gel-like precipitate was formed, and the uniform gelation of the complex was somewhat inhibited. 2. pH measurement Fig. 1 shows a system without tannic acid, and Fig. 2 shows a system with tannic acid added. As shown in these figures, the pH settles to near neutral over time. There was a tendency to come. 3. X-ray diffraction As shown in FIG. 4, a system using α-TCP (Example 3,
In (4), after 1 to 3 days, crystallization conversion to apatite was started while forming OCP, and almost one week later, it was completely replaced with apatite. System using OCP (Examples 1 and 2)
However, as shown in FIG. 3, although a little, the synthesis of apatite was confirmed. Some of these apatites are considered to be carbonate apatite. In the comparative example, as shown in FIG. 5, almost no crystal conversion was confirmed. 4. Scanning electron microscope In the OCP system (Examples 1 and 2), after one day, almost the entire surface is covered with scaly or scale-like crystals, and the host crystal surface is relatively flat. Three days and one week later, granular crystals become noticeable on the surface of the parent crystal.
No difference was observed between the presence and absence of tannic acid.

Among the systems using α-TCP, in the case of the system containing no tannic acid (Example 3), a granular and well-structured structure was observed, and a composite material was formed by entanglement of the generated crystals and a matrix of collagen. Seems to be doing.
Also, three days after the tannic acid-containing system (Example 4), a petal-like crystal structure showing typical OCP formation was observed,
Crystal conversion to OCP is evident.

In the comparative example, no crystal growth was observed. 5. In each of Examples and Comparative Examples, the composite was freeze-dried. In the tannic acid-containing system, there was a feeling of dryness and it took time to dry. On the other hand, in the case of no tannic acid addition, all of them were cotton-like materials having good flexibility. -Example 5-0.3% aqueous solution of collagen-IP (p.
H3), a 10-fold concentration solution of MEM medium and a reconstitution buffer were mixed at a ratio of 8: 1: 1. To this mixture,
The OCP dried and heated at 50 ° C. is mixed with OCP / mixture = 0.5 g /
The mixture was added at a rate of 10 ml and stirred for 1 hour. The obtained composite was dispensed into a multiplate and placed in a CO ₂ incubator (37 ° C., CO ₂ = 5%) for 1 day, 3 days, 1 week,
For 3 weeks. These were subjected to 10% formalin treatment and 100% ethanol treatment and air-dried to obtain a composite material for the purpose of analyzing crystal conversion and crystal growth.

Example 6 The mixture was 0.3% of UV-sterilized collagen-IP
Example 5 was repeated except that an aqueous solution (pH 3), a BGJ-b medium 5-fold concentration solution, and a reconstitution buffer were mixed at a ratio of 7: 2: 1. Example 7 The procedure of Example 5 was repeated, except that α-TCP sterilized by dry heat at 150 ° C. was used instead of the OCP.

Example 8 The procedure of Example 6 was repeated, except that α-TCP sterilized by dry heat at 150 ° C. was used instead of OCP. Example 9 The procedure of Example 5 was repeated, except that TeCP sterilized by dry heat at 150 ° C. was used instead of OCP.

Example 10 The procedure of Example 6 was repeated, except that TeCP sterilized by dry heat at 150 ° C. was used instead of OCP. Comparative Example 3 The procedure of Example 5 was repeated, except that instead of OCP, HAp sterilized by dry heat at 150 ° C. was used.

Comparative Example 4 The procedure of Example 6 was repeated, except that HAp sterilized by dry heat at 150 ° C. was used instead of OCP. The physical properties described above were measured for Examples 5 to 10 and Comparative Examples 3 and 4. The results are shown in FIGS. 1. pH Measurement As shown in FIGS. 6 and 7, in Examples 5 and 6 (system using OCP) and Comparative Examples 3 and 4 (system using HAp), p
H fluctuation is small and relatively stable. Examples 7 and 8 (α
In the (system using -TCP), the pH changes to the acidic side over time, but the fluctuation range is slightly smaller in Example 8 using the BGJ-b medium. Therefore, it is considered that pH fluctuation can be suppressed by selecting an appropriate medium or the like. In Examples 9 and 10 (systems using TeCP), p
H fluctuates initially, but gradually becomes neutral. 2. In the system using the X-ray diffraction OCP (Examples 5 and 6), as shown in FIGS. 8 to 11, crystal transformation with time is not so much confirmed, but as shown in FIG. Some carbonate apatite is synthesized. In the system using α-TCP (Examples 7 and 8), as shown in FIGS. As shown in FIG. 17, this conversion reaction was completed in about 1 to 2 weeks, and α-
The transition from carbonate apatite to hydroxyapatite is accompanied by a decrease in the TCP peak. Also in the system using TeCP (Examples 9 and 10), as shown in FIGS.
Crystallization over time was confirmed. This conversion reaction is shown in FIG.
As shown in FIG. 2, the reaction was completed in about 1 to 2 weeks, hydroxyl apatite was synthesized from the beginning, and the TeCP peak declined with time. On the other hand, as shown in FIGS. 23 to 27, in the comparative example, almost no crystallization over time was observed. 3. Scanning electron microscope In the system using OCP (Examples 5 and 6), the crystal surface is relatively flat, and scaly crystals are partially observed. α
In the system using -TCP (Examples 7 and 8), a petal-like structure was shown throughout, and in the long term, large granules were also confirmed.
It can be seen that CP has been synthesized. Example 8 using the BGJ-b medium shows a larger petal-like structure, and seems to have higher α-TCP activity. Also, Te
In the system using CP (Examples 9 and 10), many fine granular crystals were observed on the surface of large granules, and as in the system using α-TCP, the system in Example 10 using the BGJ-b medium was used. However, the activity of TeCP seems to be high. On the other hand, in the comparative example, the crystal structure of the surface did not change throughout.

Further, Examples 1 to 10 and Comparative Example 1
3 days after the reaction, the lyophilized material without formalin treatment was used as a test material, and a drill hole was formed in the tibia of the adult rabbit, and the hemostatic effect was visually determined. Was decalcified and a non-decalcified pathological specimen was prepared. Table 2 shows the results.

[0032]

[Table 2]

[0033]

Industrial Applicability The collagen-calcium phosphate composite material of the present invention has high bioactivity and can be expected to have a positive healing effect. Furthermore, since molding such as pressing and cutting is easy and excellent in flexibility, it can be filled into a curved surface portion. Further, the gel-like substance in the course of the hydrolysis reaction becomes a three-dimensional culture base material, and further, when freeze-dried, becomes a cotton-like or film-like implant material having good flexibility.

Further, the adhesive for living body of the present invention can promote the growth of self-repairing cells, because the adhesive itself is not a foreign substance to the living body. For this reason, incision of various organs
It is suitable for hemostasis of the resected surface, joining of blood vessels and organs, and adhesion of anastomosis.

[Brief description of the drawings]

FIG. 1 shows the pH of the composite materials of Examples 1, 3 and Comparative Example 1.
Of FIG.

FIG. 2 shows the pH of the composite materials of Examples 2, 4 and Comparative Example 2.
Of FIG.

FIG. 3 shows a chart of the composite materials of Examples 1 and 2 using an X-ray diffractometer.

FIG. 4 shows a chart of the composite materials of Examples 3 and 4 using an X-ray diffractometer.

FIG. 5 shows a chart of an X-ray diffractometer of the composite materials of Comparative Examples 1 and 2.

FIG. 6 shows the variation over time of the pH of the composite materials of Examples 5, 7, 9 and Comparative Example 3.

FIG. 7 shows the variation over time of the pH of the composite materials of Examples 6, 8, 10 and Comparative Example 4.

FIG. 8 shows a chart of an X-ray diffractometer of the composite material of Example 5 after a lapse of one day.

9 shows a chart of an X-ray diffractometer of the composite material of Example 5 after a lapse of 3 weeks. FIG.

FIG. 10 shows a chart of an X-ray diffractometer of the composite material of Example 6 after a lapse of one day.

FIG. 11 shows a chart of an X-ray diffractometer of the composite material of Example 6 after a lapse of 3 weeks.

FIG. 12 is an X-ray diffraction chart showing a change over time of the composite material of Example 5.

FIG. 13 shows a chart of the composite material of Example 7 after one day by an X-ray diffractometer.

FIG. 14 shows a chart of an X-ray diffractometer of the composite material of Example 7 after a lapse of 3 weeks.

FIG. 15 shows a chart of the composite material of Example 8 after one day by an X-ray diffractometer.

FIG. 16 shows a chart of an X-ray diffractometer of the composite material of Example 8 after a lapse of 3 weeks.

FIG. 17 is an X-ray diffraction chart showing a change over time of the composite material of Example 7.

FIG. 18 shows a chart of an X-ray diffractometer of the composite material of Example 9 after elapse of one day.

FIG. 19 shows a chart obtained by an X-ray diffractometer of the composite material of Example 9 after elapse of 3 weeks.

FIG. 20 shows a chart of the composite material of Example 10 after one day by an X-ray diffractometer.

FIG. 21 shows a chart of an X-ray diffractometer of the composite material of Example 10 after a lapse of 3 weeks.

FIG. 22 is an X-ray diffraction chart showing a change over time of the composite material of Example 9.

FIG. 23 shows a chart of the composite material of Comparative Example 3 after one day by an X-ray diffractometer.

FIG. 24 shows a chart of an X-ray diffractometer of the composite material of Comparative Example 3 after 3 weeks.

FIG. 25 shows a chart of an X-ray diffractometer of the composite material of Comparative Example 4 after one day has elapsed.

FIG. 26 shows a chart of an X-ray diffractometer of the composite material of Comparative Example 4 after 3 weeks.

FIG. 27 is an X-ray diffraction chart showing a change over time of the composite material of Comparative Example 3.

Continued on the front page (72) Inventor Tomito Sugihara 2--22 Futama, Yao-shi, Osaka Nitta Zelatin Co., Ltd. Inside the Osaka Plant (72) Inventor Yoshinori Bandai 2--22 Futama, Yao-shi, Osaka Nitta Zelatin Inside the Osaka Plant Co., Ltd. (72) Koji Nagatomi 2-22-22 Futamata, Yao City, Osaka Prefecture Inside the Nitta Gelatin Co., Ltd. Osaka Plant (56) References JP-A-63-229058 (JP, A) JP-A-1 -207072 (JP, A) JP-A-1-166676 (JP, A) JP-A-1-166762 (JP, A) International Publication 90/1341 (WO, A1)

Claims (4)

(57) [Claims] 1. A composite material comprising collagen, and at least one calcium phosphate compound having a chemical activity and source of phosphate ions is formulated selected from the group consisting of-phosphate tetracalcium and octacalcium phosphate A collagen-calcium phosphate composite material, characterized in that: 2. The collagen-calcium phosphate composite material according to claim 1, wherein the weight ratio of collagen to the calcium phosphate compound having chemical activity is collagen: calcium phosphate compound = 99.9: 0.1 to 0.7: 99.3. . 3. The collagen-calcium phosphate composite material according to claim 1, further comprising a polyphenol-based crosslinking agent. 4. A bioadhesive obtained by adding gelatin and resorcinol to the composite material according to claim 1.