JP3008034B2 - Composition for transplantation of highly concentrated crosslinked atherocollagen injectable into the body - Google Patents
Composition for transplantation of highly concentrated crosslinked atherocollagen injectable into the bodyInfo
- Publication number
- JP3008034B2 JP3008034B2 JP3113660A JP11366091A JP3008034B2 JP 3008034 B2 JP3008034 B2 JP 3008034B2 JP 3113660 A JP3113660 A JP 3113660A JP 11366091 A JP11366091 A JP 11366091A JP 3008034 B2 JP3008034 B2 JP 3008034B2
- Authority
- JP
- Japan
- Prior art keywords
- atherocollagen
- crosslinked
- collagen
- atelocollagen
- polyepoxy compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Description
【０００１】[0001]
【産業上の利用分野】本発明は、体内に注入して使用す
ることができ、そして軟組織陥凹状欠損部補正修復材と
して用いることができる、ポリエポキシ化合物により架
橋したコラ−ゲン水性懸濁液に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an aqueous collagen suspension crosslinked with a polyepoxy compound, which can be used by injecting into the body and can be used as a repair material for correcting a soft tissue concave defect. About.
【０００２】[0002]
【従来の技術】コラ−ゲンは、動物の皮膚、角膜、血
管、腱、骨などに多く分布するタンパク質であり、分子
量約３０万で、３本のポリペプチド鎖からなる螺旋構造
を持ち、１分子の長さ約３００ｎｍ、直径約１．５ｎｍ
の棒状の分子である。コラ−ゲンの分子は、ミクロフィ
ブリルと呼ばれる分子５本が束となり、かつ隣の分子に
対し各々６７ｎｍだけずれた構造を形成する。このミク
ロフィブリルの太さは約４ｎｍであり、コラ−ゲン線維
の基本単位構造である。ミクロフィブリルは多数束に集
合しフィブリルを形成し、フィブリルはさらに束となり
コラ−ゲン線維を形成する。腱などは、コラ−ゲン線維
が配列した組織であり、皮膚は、コラ−ゲン線維が絡み
合った組織である。2. Description of the Related Art Collagen is a protein widely distributed in animal skin, cornea, blood vessels, tendons, bones, etc., has a molecular weight of about 300,000, has a helical structure composed of three polypeptide chains, and has a helical structure. Molecule length about 300nm, diameter about 1.5nm
Is a rod-shaped molecule. Collagen molecules form a structure in which five molecules called microfibrils are bundled and each is shifted by 67 nm from an adjacent molecule. The thickness of the microfibrils is about 4 nm, which is the basic unit structure of collagen fibers. The microfibrils aggregate into multiple bundles to form fibrils, and the fibrils further bundle to form collagen fibers. A tendon or the like is a tissue in which collagen fibers are arranged, and a skin is a tissue in which collagen fibers are intertwined.
【０００３】線維状コラ−ゲンは分子間に架橋を有し、
この分子間架橋はコラ−ゲン分子末端に存在する三重螺
旋を組まないテロペプチドに通常存在している。アテロ
コラ−ゲンは、ペプシン等の酵素処理によりテロペプチ
ド部を消化することにより得られるものである。そし
て、コラ−ゲンの主要な抗原決定基はテロペプタイド部
に存在するため、このテロペプチド部を消化させたアテ
ロコラ−ゲンは抗原性をほとんど有さず、医用材料とし
て優れた材料である。[0003] Fibrous collagen has cross-links between molecules,
This intermolecular cross-link is usually present in non-triple-helix telopeptides present at the collagen molecule termini. Atelocollagen is obtained by digesting the telopeptide moiety by enzymatic treatment with pepsin or the like. Since the major antigenic determinant of collagen is present in the telopeptide moiety, atelocollagen obtained by digesting this telopeptide moiety has little antigenicity and is an excellent material as a medical material.
【０００４】このアテロコラ−ゲンは、これを水性液に
して、皮膚などの軟組織陥凹状欠損傷における組織欠損
部に注入することによって、組織欠損部を切開すること
なく該部の修復に用いることができる。この注入の際、
一般的に、水性液のコラ−ゲン濃度が高いほど修復効果
は向上し、皮膚***効果は持続する。また、創傷治癒お
よび美容上の観点から一カ所への大量の注入ではなく数
カ所に微量注入するのが好ましく、このためには２７Ｇ
または３０Ｇなどの細い注射針を用いる必要があり、し
たがってアテロコラ−ゲンの水性液は低粘度が要求され
る。すなわち、体内注入用のアテロコラ−ゲン水性液
は、高濃度にして、低粘度のものが要求される。[0004] The atelocollagen can be used as an aqueous liquid and used for repairing a tissue defect portion without incising the tissue defect portion by injecting it into a tissue defect portion in a soft tissue depressed defect. it can. During this injection,
In general, the higher the collagen concentration of the aqueous liquid, the higher the repair effect and the longer the skin swelling effect. From the viewpoint of wound healing and cosmetics, it is preferable to inject a small amount into several places instead of a large amount into one place.
Alternatively, it is necessary to use a thin needle such as 30G, and therefore, the aqueous liquid of atelocollagen is required to have a low viscosity. That is, the atelocollagen aqueous liquid for injection into the body is required to have a high concentration and a low viscosity.
【０００５】ところで、このような体内注入用コラ−ゲ
ンとしては、コラ−ゲン中性水溶液（特公昭６２−３７
０２０号公報）、コラ−ゲン線維の水性懸濁液（米国特
許第３９４９０７３号明細書）、グルタルアルデヒドに
て架橋したアテロコラ−ゲンの水性懸濁液（特公平１−
３６８４０号公報）などが提案されている。[0005] By the way, as such a collagen for injecting into a body, a neutral aqueous solution of collagen (Japanese Patent Publication No. Sho 62-37) has been known.
No. 020), aqueous suspensions of collagen fibers (U.S. Pat. No. 3,949,073), aqueous suspensions of atelocollagen cross-linked with glutaraldehyde (Japanese Patent Publication No.
No. 36840) has been proposed.
【０００６】これらのうち、水溶液タイプのものは、中
性で、低温において粘稠な液体である。これは、生体に
移植されると体温によりコラ−ゲン線維形成を起こし、
生体コラ−ゲンと同様のコラ−ゲン構造を構築する。こ
のため、生体内での分解・吸収は緩慢であり皮膚***効
果は高い。しかし、粘稠であるため注入が、特に高濃度
の場合には困難であるほか、一ヵ所に多量に注入する
と、緻密なコラ−ゲン構造が形成され生体自己組織の細
胞の侵潤が阻害される傾向が認められる。Among these, the aqueous solution type is a neutral, viscous liquid at a low temperature. This causes collagen fiber formation due to body temperature when implanted in a living body,
Construct a collagen structure similar to the biological collagen. Therefore, decomposition and absorption in the living body are slow, and the skin swelling effect is high. However, because of its viscousness, injection is particularly difficult at high concentrations, and when injected in large quantities in one place, a dense collagen structure is formed and the infiltration of cells of the living body's own tissue is inhibited. Tend to be observed.
【０００７】また、コラ−ゲン線維の水性懸濁液のもの
は、コラ−ゲン分子分散液を生体と等しい条件にするこ
とにより線維形成させ、水に分散したるものである。こ
のままでは細かい注射針を用いた注入による移植が困難
であるため、線維形成後に再度微細構造にしなければな
らない。これにより、注入は容易となるが、体内に移植
されたコラ−ゲンが注入直後に皮内に拡散し、かつ分解
・吸収も速やかに起こる。このため、皮膚***効果は低
く注入を頻繁に行わなければならなかった。この欠点を
改善する目的でコラ−ゲン濃度を高めたものも開発され
たが、注入するコラ−ゲンの物理的状態は変わらないた
め、体内における拡散、分解・吸収が速く皮膚***効果
の改善はあまり認められなかった。An aqueous suspension of collagen fibers is prepared by forming a collagen molecule dispersion liquid under the same conditions as a living body to form fibers and dispersing the fibers in water. It is difficult to implant by injection using a fine injection needle as it is, and it is necessary to re-fine the structure after fibril formation. This facilitates the injection, but the collagen implanted in the body diffuses into the skin immediately after the injection, and decomposition and absorption occur promptly. For this reason, the skin swelling effect was low and injection had to be performed frequently. In order to remedy this drawback, a collagen with a higher concentration has been developed.However, since the physical state of the collagen to be injected does not change, its diffusion, decomposition and absorption in the body are fast, and the improvement of the skin swelling effect is not improved. Not much.
【０００８】更に、これら未架橋のコラ−ゲン注入材の
場合、主要な抗原決定基であるテロペプタイドは除去さ
れているにもかかわらず２〜３％の人に反応が認めら
れ、かつ、反応のない人においても継続的に投与を繰り
返すと２〜３％に新たに反応が認められ、問題となる。Further, in the case of these uncrosslinked collagen injection materials, a reaction is observed in 2-3% of persons even though telopeptide which is a major antigenic determinant has been removed, and Even if the patient does not have the drug, if the administration is continuously repeated, a new reaction is observed in 2-3%, which is problematic.
【０００９】上述した欠点を改善する試みとして、グル
タルアルデヒドで架橋したアテロコラ−ゲンが使用され
た。これはアテロコラ−ゲンにグルタルアルデヒドを用
いて新たに化学的な架橋を導入したものの水性懸濁液で
あり、体内における分解・吸収については抵抗性が向上
し、かつ抗原性はさらに低下した。しかし、グルタルア
ルデヒドで架橋されたアテロコラ−ゲンは疎水性に変化
し、水性懸濁液とした場合の流動性は未架橋タイプのも
のに比べて劣り、特に高濃度においては流動性が悪くな
り注入が困難となる。さらに移植物の生体内における経
時的変化として、自己組織への同一化が低く、また石灰
化などの器質化がしばしば認められ、特に高濃度におい
ては顕著である。このため、高濃度のものを注入するこ
とは適当ではなく、皮膚***効果は未架橋のものに比
べ、いくぶん改善されたにすぎない。また架橋剤として
用いられているグルタルアルデヒドには細胞毒性があ
り、この点でも問題となる。In an attempt to ameliorate the above-mentioned disadvantages, glutaraldehyde crosslinked atherocollagens have been used. This is an aqueous suspension obtained by introducing a new chemical crosslink into atelocollagen using glutaraldehyde, and has improved resistance to degradation and absorption in the body and further reduced antigenicity. However, the atelocollagen cross-linked with glutaraldehyde changes to hydrophobic, and the fluidity of the aqueous suspension is inferior to that of the non-crosslinked type, especially at high concentrations, where the fluidity is poor. Becomes difficult. Further, as the time course of the implant in the living body, identification into self-tissue is low, and organization such as calcification is often observed, particularly at a high concentration. For this reason, it is not appropriate to inject high concentrations, and the skin bumping effect is only somewhat improved compared to the uncrosslinked one. Glutaraldehyde used as a cross-linking agent has cytotoxicity, which also poses a problem.
【００１０】[0010]
【発明が解決しようとする課題】本発明は、上記の問題
に鑑み、高濃度にして使用しても、低粘度で流動性が良
く注入が容易であり、かつ抗原性が低く、石灰化を起こ
さず、毒性のない、皮膚***効果の持続性に優れた体内
注入用架橋化アテロコラ−ゲン組成物を提供することを
目的とする。SUMMARY OF THE INVENTION In view of the above problems, the present invention has a low viscosity, has good fluidity, is easy to inject, and has a low antigenicity even when used at a high concentration. An object of the present invention is to provide a crosslinked atherocollagen composition for infusion into a body, which does not occur, has no toxicity, and has excellent persistence of skin swelling effect.
【００１１】[0011]
【問題点を解決するための手段】上記の目的を達成する
ため、本発明者らは、体内注入に使用するアテロコラ−
ゲンの架橋剤について種々検討を行なった結果、架橋剤
としてポリエポキシ化合物が極めて適することを知り、
本発明を完成するに至った。In order to achieve the above-mentioned object, the present inventors have developed an atherocolla used for infusion into a body.
As a result of conducting various studies on the cross-linking agent for gen, we found that polyepoxy compounds are extremely suitable as a cross-linking agent.
The present invention has been completed.
【００１２】即ち、本発明は、緩衝液により生理的な状
態に調整されたアテロコラ−ゲン水性懸濁液からなり、
（ａ）アテロコラ−ゲン含有量が５５〜７５ｍｇ／ｍｌ
であり、（ｂ）アテロコラ−ゲンの２０〜１００重量％
がポリエポキシ化合物で架橋された架橋アテロコラ−ゲ
ンである移植用組成物に係わる。That is, the present invention comprises an aqueous atherocollagen suspension adjusted to a physiological state by a buffer,
(A) an atherocollagen content of 55 to 75 mg / ml
(B) 20 to 100% by weight of atherocollagen
Is a crosslinked atherocollagen crosslinked with a polyepoxy compound.
【００１３】本発明で用いるアテロコラ−ゲンは、種々
の動物の結合組織に、ペプシン等の酵素を作用させ、コ
ラ−ゲン分子間の架橋を切断することにより可溶化、抽
出されるコラ−ゲンである。酵素による可溶化の際、抗
原決定基であるテロペプタイドが除去されるため、アテ
ロコラ−ゲンの供与体と移植される受容体は遺伝学的に
同一種である必要は無い。アテロコラ−ゲンは、通常、
入手の容易さにより牛の真皮をペプシンにより可溶化し
て得ている。The atelocollagen used in the present invention is a collagen which is solubilized and extracted by causing an enzyme such as pepsin to act on connective tissues of various animals to cleave the crosslinks between the collagen molecules. is there. Since the determinant telopeptide is removed during the solubilization by the enzyme, the donor of the atelocollagen and the recipient to be transplanted need not be of the same genetic species. Atherocollagen is usually
It is obtained by solubilizing bovine dermis with pepsin due to its availability.
【００１４】本発明で架橋に用いるポリエポキシ化合物
としては、親水性ポリエポキシ化合物が好ましく、特に
ポリエ−テルポリオ−ル誘導体が好ましい。たとえば、
グリセロ−ルジグリシジルエ−テル、グリセロ−ルトリ
グリシジルエ−テル、ジグリセロ−ルテトラグリシジル
エ−テル、トリグリセロ−ルペンタグリシジルエ−テ
ル、ポリ（メチレングリコ−ル）ジグリシジルエ−テル
（重合度１〜１０）、ポリ（エチレングリコ−ル）ジグ
リシジルエ−テル（重合度１〜１０）、ポリ（トリメチ
レングリコ−ル）ジグリシジルエ−テル（重合度１〜
８）、ポリ（プロピレングリコ−ル）ジグリシジルエ−
テル（重合度１〜８）などが挙げられる。これらの架橋
剤を用いる架橋反応は、通常水系にて２０℃〜３７℃の
温度で、０．５〜７２時間行なわれる。ここで言う水系
とは、蒸留水、そのｐＨを塩酸或は水酸化ナトリウムな
どにより変化させた水、またリン酸緩衝液、ホウ酸緩衝
液、炭酸緩衝液などで緩衝した水などで、特に限定され
るものではない。The polyepoxy compound used for crosslinking in the present invention is preferably a hydrophilic polyepoxy compound, and particularly preferably a polyetherpolyol derivative. For example,
Glycerol diglycidyl ether, glycerol triglycidyl ether, diglycerol tetraglycidyl ether, triglycerol pentaglycidyl ether, poly (methylene glycol) diglycidyl ether (degree of polymerization 1 to 10) ), Poly (ethylene glycol) diglycidyl ether (degree of polymerization 1 to 10), poly (trimethylene glycol) diglycidyl ether (degree of polymerization 1 to 1)
8), poly (propylene glycol) diglycidyl ether
Ter (polymerization degree: 1 to 8). The crosslinking reaction using these crosslinking agents is usually performed in an aqueous system at a temperature of 20 ° C. to 37 ° C. for 0.5 to 72 hours. The term “aqueous system” used herein means distilled water, water whose pH has been changed with hydrochloric acid or sodium hydroxide, or water buffered with a phosphate buffer, a borate buffer, a carbonate buffer, or the like. It is not something to be done.
【００１５】またポリエポキシ化合物はアテロコラ−ゲ
ン１ｇ当り０．１ｍｇ〜２ｇの割合で使用する。反応液
中のアテロコラ−ゲン濃度は０．１重量％〜５重量％の
範囲で行ないえる。所定の時間反応させた後、架橋反応
混合物を回収し、これを、グリシン、エタノ−ルアミン
などのアミノ基を有する試薬と反応させ、架橋反応を停
止させる。次いで洗浄を十分に行なった後、アテロコラ
−ゲン濃度が６重量％〜１０重量％になるまで濃縮す
る。The polyepoxy compound is used in an amount of 0.1 mg to 2 g per 1 g of atelocollagen. The concentration of atelocollagen in the reaction solution can be in the range of 0.1% by weight to 5% by weight. After reacting for a predetermined time, a cross-linking reaction mixture is recovered and reacted with a reagent having an amino group such as glycine or ethanolamine to stop the cross-linking reaction. Next, after sufficient washing, the mixture is concentrated until the concentration of atelocollagen becomes 6% by weight to 10% by weight.
【００１６】次いでこれに、リン酸緩衝液を加え、生理
的な状態に調整すると共に、アテロコラ−ゲン濃度を５
５〜７５ｍｇ／ｍｌ（５．５〜７．５重量％）に調整す
る。かくして本発明の目的とする体内注入可能な高濃度
架橋化アテロコラ−ゲン移植用組成物を得ることができ
る。Next, a phosphate buffer solution was added to the mixture to adjust it to a physiological condition, and the atelocollagen concentration was adjusted to 5%.
Adjust to 5 to 75 mg / ml (5.5 to 7.5% by weight). Thus, the high-concentration crosslinked atherocollagen transplantable composition which can be injected into the body, which is the object of the present invention, can be obtained.
【００１７】通常、上記の如くして架橋反応させると、
アテロコラ−ゲン液中の全てのアテロコラ−ゲン分子が
架橋されて架橋化アテロコラ−ゲンとなる。すなわちア
テロコラ−ゲンの１００重量％がポリエポキシ化合物で
架橋されたものが得られる。本発明の移植用組成物にお
いては、このアテロコラ−ゲン分子の全てがポリエポキ
シ化合物で架橋されたものが用いられるが、該組成物中
に存在するアテロコラ−ゲン分子の２０重量％以上、好
ましくは４０重量％以上がポリエポキシ化合物で架橋さ
れているものも使用できる。このようにするには、例え
ば上記の如く架橋化反応させたアテロコラ−ゲン液に、
架橋化アテロコラ−ゲンの含有量が所定量となるよう
に、アテロコラ−ゲンを添加して調製する。架橋化アテ
ロコラ−ゲンの含有量が２０重量％未満では、体内にお
ける分解・吸収を充分に抑制することが出来なく、軟組
織陥凹状欠損部に注入してその***効果を長期に亘り維
持することができない。Usually, when a crosslinking reaction is carried out as described above,
All atherocollagen molecules in the atherocollagen solution are crosslinked to form crosslinked atherocollagen. That is, a product obtained by crosslinking 100% by weight of atelocollagen with a polyepoxy compound is obtained. In the transplant composition of the present invention, all of the atelocollagen molecules cross-linked by a polyepoxy compound are used, but at least 20% by weight, preferably at least 20% by weight of the atelocollagen molecules present in the composition. Those having 40% by weight or more crosslinked with a polyepoxy compound can also be used. To do so, for example, the atelocollagen solution that has been cross-linked as described above is
It is prepared by adding atelocollagen such that the content of crosslinked atherocollage becomes a predetermined amount. When the content of the crosslinked atherocollagen is less than 20% by weight, the decomposition and absorption in the body cannot be sufficiently suppressed, and the protuberance can be maintained for a long time by injecting into the soft tissue depressed defect. Can not.
【００１８】また、本発明における架橋化アテロコラ−
ゲンは、アテロコラ−ゲン分子の側鎖アミノ基の１０％
以上がポリエポキシ化合物と反応している。この反応が
アミノ基の１０％未満のものは、架橋させた効果が発揮
されない。すなわち、アテロコラ−ゲンの体内における
分解・吸収を充分に抑制することが出来ない。The crosslinked atherocolla according to the present invention is
Gen is 10% of the side chain amino group of the atherocollagen molecule.
The above has reacted with the polyepoxy compound. If this reaction is less than 10% of the amino groups, the effect of crosslinking is not exhibited. That is, it is not possible to sufficiently suppress the decomposition and absorption of atelocollagen in the body.
【００１９】本発明のポリエポキシ化合物で架橋化した
アテロコラ−ゲンの水性懸濁液は、他の架橋剤による架
橋化アテロコラ−ゲンの水性懸濁液と異なり、同濃度に
おいて極めて低い粘度を示すと言う特異な挙動を示す。
例えば、グルタルアルデヒドで架橋したアテロコラ−ゲ
ンの３．５％水性懸濁液は、回転粘度計を用い粘弾性を
測定すると、回転数１００ｓ-1の剪断応力の場合５００
０ｍＰａ・ｓを示すが、ポリエポキシ化合物架橋化コラ
−ゲンの場合は、その３．５％水性懸濁液においては、
回転数１００ｓ-1の剪断応力の場合９００ｍＰａ・ｓと
いう低い粘度特性を示し、６％水性懸濁液においてさえ
回転数１００ｓ-1の剪断応力の場合５０００ｍＰａ・ｓ
である。An aqueous suspension of atherocollagen cross-linked with the polyepoxy compound of the present invention, unlike an aqueous suspension of atherocollagen cross-linked with another cross-linking agent, exhibits extremely low viscosity at the same concentration. It shows unique behavior.
For example, a 3.5% aqueous suspension of atelocollagen cross-linked with glutaraldehyde can be measured for viscoelasticity using a rotational viscometer at a rotational stress of 100 s -1 at a shear stress of 500.
Although it shows 0 mPa · s, in the case of the polyepoxy compound cross-linked collagen, in its 3.5% aqueous suspension,
It exhibits low viscosity characteristics of 900 mPa · s at a shear stress of 100 s −1 and 5000 mPa · s at a shear stress of 100 s −1 even in a 6% aqueous suspension.
It is.
【００２０】このように、本発明におけるのポリエポキ
シ化合物架橋化アテロコラ−ゲン水性懸濁液は、高濃度
においても低粘度で流動性が良いため体内への注入をス
ム−スに容易に行い得る利点がある。したがって本発明
においては、５５〜７５ｍｇ／ｍｌという高濃度におい
て体内に注入することが出来る。そして、高濃度にして
体内に注入するほど皮膚***効果が発揮される。５５ｍ
ｇ／ｍｌ未満の濃度では、皮膚***効果、その保持効果
すなわち容量保持効果が未だ充分とは言い難い。また、
７５ｍｇ／ｍｌを超える濃度になるポリエポキシ化合物
架橋化アテロコラ−ゲン水性懸濁液の粘度が高くなり、
流動性が低下するため注入が困難となる。Thus, the polyepoxy compound-crosslinked aqueous atherocollagen suspension of the present invention has a low viscosity and good fluidity even at a high concentration, so that it can be easily and smoothly injected into the body. There are advantages. Therefore, in the present invention, it can be injected into the body at a high concentration of 55 to 75 mg / ml. The higher the concentration and the more the substance is injected into the body, the more the skin swelling effect is exerted. 55m
At a concentration of less than g / ml, it is difficult to say that the skin swelling effect and its holding effect, that is, the capacity holding effect are still sufficient. Also,
Increasing the viscosity of the polyepoxy compound crosslinked atherocollagen aqueous suspension to a concentration above 75 mg / ml,
Injection becomes difficult due to reduced fluidity.
【００２１】また本発明における架橋化アテロコラ−ゲ
ンは、示差走査熱量計を用いた転移温度の測定で４０℃
以上の転移温度（ゼラチンに転移する温度）を示すもの
が好ましく、特に転移温度５０〜８０℃のものが望まし
い。転移温度が４０度未満のものは架橋の程度が充分で
なく、アテロコラ−ゲンの体内における分解・吸収を充
分に抑制することが出来ない。The crosslinked atelocollagen of the present invention has a transition temperature of 40 ° C. as measured by a differential scanning calorimeter.
Those exhibiting the above transition temperature (temperature at which gelatin is transferred) are preferable, and those having a transition temperature of 50 to 80 ° C are particularly desirable. If the transition temperature is less than 40 degrees, the degree of crosslinking is insufficient, and the decomposition and absorption of atelocollagen in the body cannot be sufficiently suppressed.
【００２２】本発明のポリエポキシ化合物架橋化アテロ
コラ−ゲン水性懸濁液は、架橋剤としてポリエポキシ化
合物を用いたので、グルタルアルデヒドなどを用いた場
合に比し毒性がなく、かつ抗原性が低く、石灰化を起こ
さないため、組織反応が小さく、線維芽細胞の進入及び
自己組織への同化が早く、皮膚***効果を早期に達成で
きる利点がある。したがって、交通事故、手術、外傷等
により生じた軟組織陥凹状欠損部に注入して使用するの
に好適である。また必要に応じこれに他の添加剤、例え
ば局所麻酔剤などを添加して使用することもできる。The aqueous suspension of polyepoxy compound-crosslinked atherocollagen of the present invention uses a polyepoxy compound as a crosslinking agent, and therefore has less toxicity and lower antigenicity than glutaraldehyde and the like. Since calcification does not occur, there is an advantage that tissue reaction is small, fibroblast invasion and assimilation into self-tissue is fast, and skin swelling effect can be achieved at an early stage. Therefore, it is suitable to be used by injecting it into a soft tissue recessed defect caused by a traffic accident, surgery, trauma or the like. If necessary, other additives, for example, a local anesthetic may be added and used.
【００２３】[0023]
【実施例】以下、実施例にもとづき本発明を詳細に説明
する。新鮮な仔牛の背部の皮を切り取り、その周縁部を
切り落した後、この背部の皮を水道水で洗浄して皮の外
部に付着している汚れを落し、さらにパイロジエンフリ
−水で洗浄した。こうして得られた皮を７０％エタノ−
ルに浸漬した後、カミソリを用いて毛根部を残さないよ
うに、毛及び皮の上面を削ぎ落した。この時新たに現れ
た真皮の表面に汚れが付着しないように注意する。一
方、皮の裏面もカミソリで削ぎ落し、こうして仔牛皮の
真皮層のみを、汚染されないように取り出した。この真
皮層を７０％アルコ−ルに一夜浸漬後、過剰のアルコ−
ルを除去した後、無菌的に粉砕した。次にパイロジエン
フリ−の５％ＮａＣｌ水で洗浄し、遠心脱水し、さらに
パイロジエンフリ−水で洗浄後、７０％アルコ−ルに一
夜浸漬した。遠心脱液により過剰のアルコ−ルを除去
し、粉砕真皮を無菌の溶解槽に入れ、パイロジエンフリ
−水を加え、さらに、パイロジエンフリ−水に溶解し且
つ濾過除菌したペプシンを加えた。この際、ペプシンは
粉砕真皮に対し０．５％（乾燥重量基準で）を加えた。
また、粉砕真皮の濃度を約０．５％〜０．９％、ｐＨを
ＨＣｌにより３に調整し、温度を２０℃に維持した。こ
の溶解槽内の混合物を２０℃で３日間緩やかに撹拌処理
し、真皮不溶性コラ−ゲンを完全に溶解して、アテロコ
ラ−ゲン溶液を得た。DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail based on embodiments. The skin of the back of a fresh calf was cut off, the periphery was cut off, and the skin on the back was washed with tap water to remove the dirt attached to the outside of the skin, and further washed with pyridien-free water. . 70% ethanol-
After being immersed in the hair, the upper surface of the hair and skin was scraped off using a razor so as not to leave the root of the hair. At this time, care should be taken so that dirt does not adhere to the newly appearing dermis surface. On the other hand, the back of the skin was also shaved off with a razor, and only the dermal layer of the calf skin was taken out without contamination. After immersing this dermis layer in 70% alcohol overnight, excess alcohol was added.
And then aseptically pulverized. Next, it was washed with 5% NaCl aqueous solution of pyridien free, centrifugally dehydrated, further washed with water containing pyridien free, and immersed in 70% alcohol overnight. Excess alcohol was removed by centrifugal drainage, and the ground dermis was placed in a sterile dissolving tank, to which was added pyrogen-free water, and then pepsin which had been dissolved in pyridien-free water and filtered and sterilized was added. . At this time, 0.5% (based on dry weight) of pepsin was added to the ground dermis.
The concentration of the ground dermis was adjusted to about 0.5% to 0.9%, the pH was adjusted to 3 with HCl, and the temperature was maintained at 20 ° C. The mixture in the dissolving tank was gently stirred at 20 ° C. for 3 days to completely dissolve the dermis-insoluble collagen to obtain an atherocollagen solution.
【００２４】ペプシン可溶化仔牛真皮由来アテロコラ−
ゲン水溶液（ｐＨ３）を、滅菌した孔径１μｍ、０．８
０μｍ、０．６５μｍ及び０．４５μｍのフィルタ−を
用いて順次濾過し、０．４５μｍのフィルタ−について
は２回濾過した。この溶液に無菌０．５Ｎ−ＮａＯＨを
加えｐＨ７に調整し線維形成を行い、パイロジエンフリ
−、２段蒸留水を用い、２％−アテロコラ−ゲン水溶液
を調整した。これに０．０４Ｍ−Ｎａ2ＨＰＯ4、０．３
Ｍ−ＮａＣｌ水溶液を加え、ｐＨ９とし、３５℃にて５
時間保温撹拌した後、エチレングリコ−ルジグリシジル
エ−テルを最終濃度を０．５％となるまで加えた。３５
℃にて１５時間撹拌しながら反応させ、反応終了後分散
液を遠心分離にて回収した。Atelocolla from pepsin-solubilized calf dermis
Gen. aqueous solution (pH 3) was sterilized with a pore size of 1 μm, 0.8 μm
Filtration was performed sequentially using a 0 μm, 0.65 μm, and 0.45 μm filter, and a 0.45 μm filter was filtered twice. Sterile 0.5N-NaOH was added to this solution to adjust the pH to 7, fibrillation was performed, and a 2% aqueous solution of atherocollagen was prepared using pyrodiene free and double distilled water. 0.04M-Na 2 HPO 4 , 0.3
An aqueous solution of M-NaCl was added to adjust the pH to 9, and the solution
After heating and stirring for hours, ethylene glycol diglycidyl ether was added until the final concentration was 0.5%. 35
The reaction was carried out while stirring at 15 ° C. for 15 hours. After the reaction was completed, the dispersion was recovered by centrifugation.
【００２５】得られた反応生成物を二段蒸留水に分散し
てコラ−ゲン濃度を２％となし、さらに０．０４Ｍ−Ｎ
ａ2ＨＰＯ4、０．３Ｍ−ＮａＣｌ、０．４Ｍ−グリシン
水溶液を加え、コラ−ゲン濃度１％に調整し、３５℃に
て１５時間撹拌し、未反応のエポキシ基を失活させた。
これを遠心分離にて回収、０．０２Ｍ−Ｎａ2ＨＰＯ4、
０．１５Ｍ−ＮａＣｌ水溶液にて十分に洗浄した後、コ
ラ−ゲン濃度６重量％（６０ｍｇ／ｍｌ）のポリエポキ
シ化合物架橋化アテロコラ−ゲン水性懸濁液を得た。こ
の架橋化アテロコラ−ゲンのε−アミノ基修飾率をＴＮ
ＢＳ法にて調べたところ、４９．２％であり、示差走査
熱量計による分析で転移温度６０．１℃を示した。The obtained reaction product was dispersed in double-distilled water to obtain a collagen concentration of 2%, and 0.04 M-N
a 2 HPO 4 , 0.3M-NaCl, 0.4M-glycine aqueous solution was added to adjust the collagen concentration to 1%, and stirred at 35 ° C. for 15 hours to deactivate unreacted epoxy groups.
This was collected by centrifugation, and 0.02 M-Na 2 HPO 4 ,
After sufficiently washing with an aqueous 0.15 M NaCl solution, an aqueous suspension of polyepoxy compound crosslinked atelocollagen having a collagen concentration of 6% by weight (60 mg / ml) was obtained. The ε-amino group modification rate of this crosslinked atherocollagen was determined by TN
It was 49.2% when examined by the BS method, and showed a transition temperature of 60.1 ° C. by analysis with a differential scanning calorimeter.
【００２６】粘弾性の測定 東京計器製コ−ンプレ−トタイプＥ型回転粘度計を用い
測定した。結果を図１、図２および図３に示す。図１は
エチレングリコ−ルジグリシジルエ−テル架橋化アテロ
コラ−ゲンの３．５重量％（３５ｍｇ／ｍｌ）水性懸濁
液の粘弾測定結果で、回転数１００ｓ-1の剪断応力のと
き９００ｍＰａ・ｓという低い粘度特性を示し、また図
２にみるように、６重量％（６０ｍｇ／ｍｌ）水性懸濁
液のとき回転数１００ｓ-1の剪断応力で５０００ｍＰａ
・ｓを示した。図３は従来のグルタルアルデヒドで架橋
したアテロコラ−ゲンの３．５重量％（３５ｍｇ／ｍ
ｌ）水性懸濁液の粘弾測定結果で、このような低濃度に
おいてさえ、回転数１００ｓ-1の剪断応力で５０００ｍ
Ｐａ・ｓを示した。このように、本発明のポリエポキシ
化合物架橋化アテロコラ−ゲン水性懸濁液は、他の架橋
剤による架橋化アテロコラ−ゲン水性懸濁液と異なり極
めて低い粘度を示した。Measurement of viscoelasticity It was measured using a composite plate type E type viscometer manufactured by Tokyo Keiki. The results are shown in FIGS. 1, 2 and 3. FIG. 1 shows the results of viscoelasticity measurement of an aqueous suspension of 3.5% by weight (35 mg / ml) of ethylene glycol diglycidyl ether crosslinked atelocollagen at 900 mPa · s at a rotational stress of 100 s −1 and a shear stress of 100 m −1. show low viscosity properties of s, and as seen in FIG. 2, 6 wt% (60mg / ml) 5000mPa at a shear stress of rotational speed 100s -1 when the aqueous suspension
・ Indicated s. FIG. 3 shows 3.5% by weight (35 mg / m 2) of the conventional glutaraldehyde-crosslinked atelocollagen.
l) Viscoelastic measurements of the aqueous suspension show that even at such low concentrations, 5000 m at a shear rate of 100 s -1
Pa · s was indicated. As described above, the aqueous suspension of polyepoxy compound crosslinked atherocollagen of the present invention exhibited an extremely low viscosity unlike the aqueous suspension of crosslinked atherocollagen by another crosslinking agent.
【００２７】ｉｎ．ｖｉｔｒｏ試験 ヒト線維芽細胞による培養試験によって、ポリエポキシ
化合物架橋化コラ−ゲンの細胞増殖を試験した。培養皿
に、ポリエポキシ化合物架橋化コラ−ゲンを塗布し、風
乾後、培養皿に６．５×１０4個の細胞を播いて３７℃
のＣＯ2インキュベ−タを用い培養した。又比較のため
未架橋のコラ−ゲン及びグルタルアルデヒド架橋化コラ
−ゲンを用いて同様に培養した。結果を第４図に示す。
図４において、黒丸はポリエポキシ化合物架橋化アテロ
コラ−ゲン、白四角は未架橋アテロコラ−ゲン、黒三角
はグルタルアルデヒド架橋化アテロコラ−ゲンについて
のそれぞれの結果である。上記ポリエポキシ化合物架橋
化アテロコラ−ゲンを塗布したものは細胞が良好な増殖
を示し、細胞の形態も正常な形態を有していた。一方、
未架橋アテロコラ−ゲン及びグルタルアルデヒド架橋化
アテロコラ−ゲンを塗布したものは細胞の増殖が見られ
なかった。In. In vitro test The cell growth of polyepoxy compound cross-linked collagen was tested by a culture test with human fibroblasts. A polyepoxy compound cross-linked collagen was applied to a culture dish, air-dried, and then 6.5 × 10 4 cells were seeded on the culture dish at 37 ° C.
Using a CO 2 incubator. For comparison, the same culture was carried out using uncrosslinked collagen and glutaraldehyde crosslinked collagen. The results are shown in FIG.
In FIG. 4, the closed circles indicate the results for the polyepoxy compound crosslinked atherocollagen, the open squares indicate the results for the uncrosslinked atherocollagen, and the closed triangles indicate the results for the glutaraldehyde crosslinked atherocollagen. The cells coated with the polyepoxy compound-crosslinked atherocollage showed good growth of cells and normal cell morphology. on the other hand,
The cells coated with uncrosslinked atelocollagen and glutaraldehyde crosslinked atelocollagen showed no cell proliferation.
【００２８】ｉｎ．ｖｉｖｏ試験 ４週齢のオスのＳＤ−ラットを用い、背部皮下に上記調
製した６重量％（６０ｍｇ／ｍｌ）ポリエポキシ化合物
架橋化アテロコラ−ゲン水性懸濁液０．３ｍｌを注入し
た。一定期間毎に生検し、組織学的に検討した。結果を
表１に示す。また、比較のため、３．５重量％（３５ｍ
ｇ／ｍｌ）ポリエポキシ化合物架橋化アテロコラ−ゲン
水性懸濁液０．３ｍｌを注入したときの結果を表２に示
す。なお、各表中（−）は認めない、少ない、（±）は
正常、普通、（＋）はやや多い、(＋＋)は多い、大き
い、(＋＋＋)激しい、極めて大きい、を示す。ポリエポ
キシ化合物架橋化アテロコラ−ゲン水性懸濁液の濃度が
高いほど容量保持効果が良いことがわかる。In. In vivo test Using a 4-week-old male SD-rat, 0.3 ml of the aqueous suspension of 6 wt% (60 mg / ml) polyepoxy compound-crosslinked atherocollagen prepared above was injected subcutaneously into the back. Biopsies were taken at regular intervals and examined histologically. Table 1 shows the results. For comparison, 3.5% by weight (35 m
g / ml) Table 2 shows the results when 0.3 ml of the aqueous suspension of polyepoxy compound crosslinked atherocollagen was injected. In each of the tables, (-) is not recognized, small, (±) is normal, (+) is slightly large, (++) is large, large, (++) severe, and extremely large. It can be seen that the higher the concentration of the aqueous suspension of polyepoxy compound crosslinked atelocollagen, the better the capacity retention effect.
【００２９】[0029]
【表１】 [Table 1]
【００３０】[0030]
【表２】 [Table 2]
【００３１】[0031]
【発明の効果】本発明の体内注入用架橋化アテロコラ−
ゲン組成物は、アテロコラ−ゲンの体内吸収性を制御す
るため架橋化する架橋剤としてポリエポキシ化合物を用
いたので、高濃度で使用しても、低粘度で流動性が良
く、体内注入をスム−スに行なうことができ、したがっ
て***効果を長期に亘り維持できる。しかも本発明の体
内注入用架橋化アテロコラ−ゲン組成物は、毒性が無
く、かつ抗原性が低く、石灰化を起こさないため、組織
反応が小さく、線維芽細胞の進入及び自己組織への同化
が早く、皮膚***効果を早期に達成できると言う顕著な
効果を奏する。EFFECT OF THE INVENTION The crosslinked atherocolla for injection into the body of the present invention.
The gen composition uses a polyepoxy compound as a cross-linking agent for cross-linking in order to control the absorption of atelocollagen into the body. And the protruding effect can be maintained over a long period of time. Moreover, the crosslinked atherocollagen composition for injection into the body of the present invention has no toxicity, low antigenicity, and does not cause calcification, so that the tissue reaction is small, and the invasion of fibroblasts and the assimilation into autologous tissues are suppressed. It has a remarkable effect that the skin swelling effect can be achieved early.
【図１】ポリエポキシ化合物架橋化アテロコラ−ゲンの
粘弾性特性図FIG. 1 is a viscoelasticity diagram of a polyepoxy compound crosslinked atherocollagen
【図２】ポリエポキシ化合物架橋化アテロコラ−ゲンの
粘弾性特性図FIG. 2 is a viscoelasticity diagram of a polyepoxy compound crosslinked atherocollagen
【図３】従来のグルタルアルデヒド架橋アテロコラ−ゲ
ンの粘弾性特性図FIG. 3 is a viscoelastic characteristic diagram of a conventional glutaraldehyde-crosslinked atherocollagen.
【図４】ポリエポキシ化合物架橋化アテロコラ−ゲンに
ついての細胞培養増殖試験の結果を示した図。FIG. 4 is a view showing the results of a cell culture growth test for a polyepoxy compound crosslinked atherocollagen.
Claims (2)
テロコラ−ゲン水性懸濁液からなり、（ａ）アテロコラ
−ゲン含有量が５５〜７５ｍｇ／ｍｌであり、（ｂ）ア
テロコラ−ゲンの２０〜１００重量％がポリエポキシ化
合物で架橋された架橋アテロコラ−ゲンであることを特
徴とする移植用組成物。1. An aqueous suspension of atherocollagen adjusted to a physiological state by a buffer, wherein (a) the content of atherocollagen is 55 to 75 mg / ml, and (b) the atherocollagen content is An implantable composition, wherein 20 to 100% by weight is a crosslinked atherocollagen crosslinked with a polyepoxy compound.
の側鎖アミノ基の１０％以上がポリエポキシ化合物と架
橋反応したものであり、示差走査熱量計による転移温度
測定において、４０℃以上の転移温度を有する請求項１
記載の移植用組成物。2. The crosslinked atherocollagen is one in which 10% or more of the side chain amino groups of the collagen molecule have undergone a crosslinking reaction with a polyepoxy compound, and has a transition temperature of 40 ° C. or more measured by a differential scanning calorimeter. 2. The method of claim 1, wherein the material has a transition temperature.
The composition for implantation according to the above.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3113660A JP3008034B2 (en) | 1990-04-23 | 1991-04-19 | Composition for transplantation of highly concentrated crosslinked atherocollagen injectable into the body |
| US07/872,722 US5314874A (en) | 1991-04-19 | 1992-04-15 | Intracorporeally injectable composition for implanting highly concentrated cross-linked atelocollagen |
| EP92303469A EP0509833B1 (en) | 1991-04-19 | 1992-04-16 | An intracorporeally injectable composition for implanting highly concentrated cross-linked atelocollagen |
| DE69212203T DE69212203T2 (en) | 1991-04-19 | 1992-04-16 | Intracorporeal injectable composition for implanting highly concentrated cross-linked atelocollagen |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2-105430 | 1990-04-23 | ||
| JP10543090 | 1990-04-23 | ||
| JP3113660A JP3008034B2 (en) | 1990-04-23 | 1991-04-19 | Composition for transplantation of highly concentrated crosslinked atherocollagen injectable into the body |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04227265A JPH04227265A (en) | 1992-08-17 |
| JP3008034B2 true JP3008034B2 (en) | 2000-02-14 |
Family
ID=26445715
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3113660A Expired - Fee Related JP3008034B2 (en) | 1990-04-23 | 1991-04-19 | Composition for transplantation of highly concentrated crosslinked atherocollagen injectable into the body |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3008034B2 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5162430A (en) * | 1988-11-21 | 1992-11-10 | Collagen Corporation | Collagen-polymer conjugates |
| US5475052A (en) * | 1988-11-21 | 1995-12-12 | Collagen Corporation | Collagen-synthetic polymer matrices prepared using a multiple step reaction |
| US5510418A (en) * | 1988-11-21 | 1996-04-23 | Collagen Corporation | Glycosaminoglycan-synthetic polymer conjugates |
| US5306500A (en) * | 1988-11-21 | 1994-04-26 | Collagen Corporation | Method of augmenting tissue with collagen-polymer conjugates |
| US5800541A (en) * | 1988-11-21 | 1998-09-01 | Collagen Corporation | Collagen-synthetic polymer matrices prepared using a multiple step reaction |
| US5565519A (en) * | 1988-11-21 | 1996-10-15 | Collagen Corporation | Clear, chemically modified collagen-synthetic polymer conjugates for ophthalmic applications |
| WO2004082694A1 (en) * | 2003-03-20 | 2004-09-30 | Cosmotec Co. Ltd. | Cell therapy material and intravascular therapy method |
| WO2006034128A2 (en) | 2004-09-17 | 2006-03-30 | Angiotech Biomaterials Corporation | Multifunctional compounds for forming crosslinked biomaterials and methods of preparation and use |
-
1991
- 1991-04-19 JP JP3113660A patent/JP3008034B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH04227265A (en) | 1992-08-17 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |