JPH05184661A - Artificial skin - Google Patents

Artificial skin

Info

Publication number
JPH05184661A
JPH05184661A JP3261753A JP26175391A JPH05184661A JP H05184661 A JPH05184661 A JP H05184661A JP 3261753 A JP3261753 A JP 3261753A JP 26175391 A JP26175391 A JP 26175391A JP H05184661 A JPH05184661 A JP H05184661A
Authority
JP
Japan
Prior art keywords
collagen
skin
artificial skin
layer
matrix
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.)
Pending
Application number
JP3261753A
Other languages
Japanese (ja)
Inventor
Mikio Koide
幹夫 小出
Risako Matsui
理佐子 松井
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Terumo Corp
Original Assignee
Terumo Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Terumo Corp filed Critical Terumo Corp
Priority to JP3261753A priority Critical patent/JPH05184661A/en
Publication of JPH05184661A publication Critical patent/JPH05184661A/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin

Abstract

PURPOSE:To provide an artificial skin which protects a skin damage part softly without drying, and replaces the part with the own tissue easily, when the skin is damaged. CONSTITUTION:A damage contact layer 2 which consists of a matrix of a denatured collagen including 0 to 80% of fibrous collagen and helix, a holding layer 3 which consists of a fabric collagen matrix with a crosslinking structure, and a steam permeation control layer 4 are laminated in order, so as to form an artificial skin 1, and a phosphoric acid ester of ascorbic acid is contained in at least either one layer. Consequently, a close adhesive property to the damaged part is improved, it can be replaced with the own tissue easily, and furthermore, after maintaining it for a specific period, a sublayer skin graft of the own body can be planted alive.

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【産業上の利用分野】本発明は、新規な人工皮膚に関す
る。さらに詳述すれば本発明は、創傷、熱傷などにより
皮膚が損傷を受けた際、該皮膚損傷部に適用され、該皮
膚損傷部を乾燥させず柔らかく保護し、早期に自己の組
織と置換する人工皮膚に関する。FIELD OF THE INVENTION The present invention relates to a novel artificial skin. More specifically, the present invention, when the skin is damaged by a wound, a burn, etc., is applied to the damaged skin part, protects the damaged skin part softly without drying, and replaces it with own tissue at an early stage. Regarding artificial skin.

【０００２】[0002]

【従来の技術】熱傷、採皮創、皮膚剥削創または外傷性
皮膚欠損創等の疾患ないし創傷による患部を保護し、治
癒を促進する目的のために、患部に一時的に適応する創
傷被覆材として、従来、ガーゼ、脱脂綿等が用いられて
いたが、これは細菌感染防止性が低く、かつ滲出液を速
やかに吸収するために創面が乾燥してしまい取りはずす
際に痛み、出血等を伴うものであった。2. Description of the Related Art A wound dressing material that is temporarily applied to an affected area for the purpose of protecting the affected area due to a disease or wound such as a burn, a skin-wound, a skin-exfoliated wound or a traumatic skin defect and promoting healing. Conventionally, gauze, absorbent cotton, etc. have been used, but this has low bacterial infection preventive property, and the wound surface is dry because it absorbs exudate promptly, and it causes pain and bleeding when removed. Met.

【０００３】また、これらに代わるものとして、特に創
面が広範囲にわたる場合に適用されるものとして、シリ
コーン製ガーゼ、シリコーンゴム製およびベロアー状の
表面構造を有するナイロン、テフロンなどの合成線維シ
ート等の人工材料の被覆膜や、凍結乾燥豚皮、フィブリ
ン膜、コラーゲン膜、ポリアミノ酸スポンジ、ムコ多糖
類複合コラーゲン膜等の生体由来材料の被覆膜も知られ
ている。しかしながら、これらのうち人工被覆膜は患部
との密着性、水蒸気透過性、ひび割れ等の点で種々の問
題を残すものであり、一方、生体由来材料の被覆膜は生
体適合性等の特徴を有するが、多くは抗原性を有し、ま
た細菌感染、滲出液による劣化などの欠点を持ち、さら
に原料が入手しにくい等の問題もあった。As an alternative to these, particularly when applied to a wide range of wound surfaces, synthetic gauze made of silicone, synthetic rubber sheet made of silicone rubber or velor, synthetic fiber sheet such as Teflon, etc. Coating films of materials and coating films of bio-derived materials such as freeze-dried pig skin, fibrin film, collagen film, polyamino acid sponge and mucopolysaccharide complex collagen film are also known. However, among these, the artificial coating film leaves various problems in terms of adhesion with the affected area, water vapor permeability, cracking, etc., while the coating film of a bio-derived material has characteristics such as biocompatibility. However, many of them have antigenicity, and also have drawbacks such as bacterial infection and deterioration due to exudate, and there is a problem that raw materials are difficult to obtain.

【０００４】さらに最近では、上記問題点を解決する方
法としてコラーゲン処理されたナイロンメッシュとシリ
コーン膜からなる複合膜が開発され、一部実用化されて
いる。該複合膜は、創面によく密着し、適度な水分透過
性を有するが、創面に固着し、肉芽組織が被覆膜中に入
り込むという欠点があった。More recently, as a method for solving the above problems, a composite membrane composed of a collagen-treated nylon mesh and a silicone membrane has been developed and partially put into practical use. The composite membrane adheres well to the wound surface and has an appropriate water permeability, but has the drawback that it adheres to the wound surface and granulation tissue penetrates into the coating film.

【０００５】また同様に、上記問題点を解決する方法と
してコラーゲンを用いた人工材料が開発されている。一
般にコラーゲン自体は生体由来材料であるため、細胞、
組織に対する親和性が大きく、生体適合性に優れ好適な
材料であることは先述したとおりであるが、反面、コラ
ーゲンは生体内で容易に分解、吸収される欠点を有する
ものである。そこで人工材料としてコラーゲンを利用す
るにあたっては適当な方法で架橋を導入し、物性面の強
化を図る工夫が成されたものが用いられている。該架橋
法としては、加熱による脱水架橋、薬品を用いる化学架
橋等を採用したものが開発されている。このうち熱脱水
架橋は薬品処理に比べ安全性が高いが、物理的にコラゲ
ナーゼ、酵素に対する耐性が化学的架橋に対し低いた
め、通常、化学架橋を熱架橋と併用させたり、または化
学的架橋単独で用いる方法が選択されている。こうした
架橋を導入したコラーゲンを用いた人工材料の場合、上
記以外に物性面で性質が著しく向上する等の優れた効果
を奏するものである。例えば１１０℃の温度で真空下に
２４時間置いて熱的な架橋を導入した場合、該人工材料
の検体ではコラゲナーゼ３ｕｎｉｔ／ｍｌ中に３７℃下
で静置すると１日以内に該人工材料中のコラーゲンが溶
解するのに対し、イソシアネート系の薬品を用いた化学
的架橋のみを施した該人工材料の検体ではコラゲナーゼ
１００ｕｎｉｔ／ｍｌ中に３７℃下で７日経過しても形
態に変化が見られない。しかしながら、前記人工材料を
人工皮膚として使用する場合、強固な架橋を導入する
と、導入前にコラーゲンが有していた細胞、組織に対す
る親和性が大幅に低下し、コラーゲンマトリックス内に
マクロファージ、好中球などの炎症性細胞や、線維芽細
胞はほとんど侵入しないまま、再生表皮のいわゆるｄｏ
ｗｎ ｇｒｏｗｔｈにより、マトリックスが排除される
欠点を有する。一方、細胞侵入性の良好なコラーゲン−
変性コラーゲンマトリックスを形成してなる前記コラー
ゲンを用いた人工材料も開発されており、早期に好中球
やマクロファージが浸潤し、さらに線維芽細胞が侵入す
ることができる。しかしながら該人工材料では開放創と
同様に創収縮を生じる欠点を有している。つまり、これ
らの人工材料においては、物性面の強化と細胞、組織に
対する親和性という生物学的性能の向上とは両立が困難
な相反する事象であり、双方を満足する創傷被覆材ある
いは人工皮膚の出現が強く望まれていた。Similarly, an artificial material using collagen has been developed as a method for solving the above problems. Generally, collagen itself is a biological material, so cells,
As described above, the material has a high affinity for tissues and is excellent in biocompatibility and is suitable, but on the other hand, collagen has a drawback that it is easily decomposed and absorbed in a living body. Therefore, when using collagen as an artificial material, a material that is devised to strengthen the physical properties by introducing crosslinks by an appropriate method is used. As the cross-linking method, those adopting dehydration cross-linking by heating, chemical cross-linking using chemicals, etc. have been developed. Of these, thermal dehydration cross-linking is safer than chemical treatment, but because physical resistance to collagenase and enzyme is physically lower than chemical cross-linking, chemical cross-linking is usually used in combination with heat cross-linking or chemical cross-linking alone. The method used in is selected. In the case of an artificial material using such a cross-linked collagen, in addition to the above, an excellent effect such as a markedly improved physical property is exhibited. For example, when thermal cross-linking is introduced by placing in a vacuum at a temperature of 110 ° C. for 24 hours, the specimen of the artificial material shows that the collagenase in the unit of 3 units / ml is kept at 37 ° C. within 1 day. In contrast to the dissolution of collagen, the specimen of the artificial material, which was only chemically crosslinked using an isocyanate-based chemical, showed a change in morphology in collagenase 100 unit / ml at 37 ° C for 7 days. Absent. However, when the artificial material is used as an artificial skin, if a strong cross-link is introduced, the affinity of collagen prior to the introduction for cells and tissues is significantly reduced, and macrophages and neutrophils are present in the collagen matrix. Such as inflammatory cells and fibroblasts hardly enter the so-called do of regenerated epidermis.
The wn growth has the drawback of eliminating the matrix. On the other hand, collagen with good cell invasion
An artificial material using the above-mentioned collagen formed by forming a denatured collagen matrix has also been developed, and neutrophils and macrophages can infiltrate early and fibroblasts can invade. However, the artificial material has the drawback of causing wound contraction as in an open wound. In other words, in these artificial materials, it is a contradictory event that it is difficult to achieve compatibility between enhancement of physical properties and improvement of biological performance such as affinity for cells and tissues. The appearance was strongly desired.

【０００６】現在までに、上記の問題点を解決する方法
として、細胞侵入性の良好なコラーゲンマトリックス層
である創傷接触層と架橋を十分に施したコラーゲンの支
持層と、水蒸気透過調節層からなる３層構造の人工皮膚
が本発明者らにより開発されている（特開平２−３４１
６５号）。この人工皮膚は動物実験では創面に適用され
た際に線維芽細胞が早期に創傷接触層に侵入し、真皮様
の結合組織を構築するので創傷の治癒が促進されてなる
ものである。この人工皮膚を広範囲の熱傷創や広範囲の
皮膚欠損部位に適用する場合、表皮の伸展等に限度があ
るため、治癒するのに非常に時間がかかる欠点を有して
いた。しかし、この欠点に関しては、一般に臨床で行わ
れている分層植皮を併用することが可能であり、ある一
定の期間上記の人工皮膚を移植した後、水蒸気透過調節
層と架橋を十分に行った支持層を剥がし、分層植皮を行
うと生着することができ、該方法により上記欠点を補う
ことができ広範囲の欠損部位に適用することも一定条件
下で可能ではある。しかし、この方法では分層植皮の移
植時期が遅くなると、コラーゲンが徐々に分解すること
から、新たに創面の拘縮がおこるため、自己のコラーゲ
ン線維の産生が必要となってくる等の課題を有してお
り、なお完全なものとすることはできず、これらの問題
点を十分に解決してなる創傷被覆材および人工皮膚は今
だ得られていないのが現状である。[0006] To date, as a method for solving the above-mentioned problems, a wound contact layer which is a collagen matrix layer having a good cell-penetrating property, a collagen cross-linking support layer having sufficient cross-linking, and a water vapor permeation control layer have been used. An artificial skin having a three-layer structure has been developed by the present inventors (JP-A-2-341).
No. 65). In animal experiments, when artificial skin is applied to the wound surface, fibroblasts infiltrate into the wound contact layer at an early stage to construct connective tissue like dermis, thereby promoting wound healing. When this artificial skin is applied to a wide range of burn wounds or a wide range of skin defect sites, it has a drawback that it takes a very long time to heal because there is a limit to the extension of the epidermis. However, regarding this drawback, it is possible to use a split-thickness skin graft that is generally performed clinically, and after transplanting the artificial skin described above for a certain period of time, sufficient cross-linking with a water vapor transmission control layer was performed. When the support layer is peeled off and split-thickness skin grafting is performed, engraftment can be achieved, the above-mentioned defects can be compensated by this method, and application to a wide range of defect sites is also possible under certain conditions. However, with this method, when the transplantation time of split-thickness skin graft is delayed, collagen gradually decomposes, and new contraction of the wound surface occurs, so it is necessary to produce self collagen fibers. The present situation is that a wound dressing and an artificial skin which have the above problems and cannot be perfected yet and have sufficiently solved these problems have not yet been obtained.

【０００７】[0007]

【発明が解決しようとする課題】したがって、本発明の
目的は、（１）創部に適度に密着して感染、痛みの防止
を図り、（２）創面に適度な湿りを与えるために、適度
な水蒸気透過性と滲出液吸収性を有し、（３）早期に自
己の良好な肉芽組織が再生され、（４）体液中のタンパ
ク質成分の漏出を防止し、（５）原料が容易に入手で
き、製造が容易であるとする要件を満たす人工皮膚を提
供することである。SUMMARY OF THE INVENTION Therefore, the object of the present invention is to (1) properly adhere to the wound to prevent infection and pain, and (2) to provide appropriate moistening to the wound surface. It has water vapor permeability and exudate absorbability, (3) it regenerates its own good granulation tissue at an early stage, (4) prevents the leakage of protein components in body fluids, and (5) makes it easy to obtain raw materials. , To provide artificial skin that meets the requirements of being easy to manufacture.

【０００８】[0008]

【課題を解決するための手段】本発明者らは、上記諸目
的を解決すべく鋭意検討した結果、細胞侵入性の良好な
コラーゲン−変性コラーゲンマトリックスによりなる創
傷部接触層がコラーゲンを骨格として、変性コラーゲン
を熱的な脱水架橋により結合させた人工皮膚により、上
記目的が達成されることを知り、この知見に基づいて本
発明を完成するに至った。Means for Solving the Problems As a result of intensive studies to solve the above-mentioned various objects, the present inventors have shown that the wound contact layer made of collagen-denatured collagen matrix having good cell-penetrating property has collagen as a skeleton, It was found that the above objects can be achieved by artificial skin in which denatured collagen is bonded by thermal dehydration crosslinking, and the present invention has been completed based on this finding.

【０００９】すなわち、本発明は、（１）線維化コラー
ゲンおよびヘリックス含量が０〜８０％である変性コラ
ーゲンのマトリックスからなる創傷接触層と、架橋構造
を有する線維化コラーゲンマトリックスからなる支持層
と、水蒸気透過調節層とが順次積層されてなり、少なく
ともいずれか１層がアスコルビン酸リン酸エステルを含
んでいる人工皮膚によって達成される。That is, the present invention provides (1) a wound contact layer comprising a matrix of fibrotic collagen and denatured collagen having a helix content of 0 to 80%, and a support layer comprising a fibrotic collagen matrix having a crosslinked structure. This is achieved by artificial skin in which a water vapor transmission control layer is sequentially laminated, and at least one of the layers contains ascorbic acid phosphate.

【００１０】本発明はまた、（２）線維化コラーゲンと
ヘリックス含量が０〜８０％である変性コラーゲンとア
スコルビン酸リン酸エステルとからなるマトリックスの
支持層とその上に積層された水蒸気透過調節層とからな
ることを特徴とする人工皮膚によって達成される。The present invention also provides (2) a matrix support layer composed of fibrillated collagen, denatured collagen having a helix content of 0 to 80%, and ascorbic acid phosphate, and a water vapor permeation control layer laminated thereon. It is achieved by artificial skin characterized by consisting of.

【００１１】本発明はさらに、（３）前記変性コラーゲ
ンの含有比が、全体の５〜３０重量％である上記（１）
または（２）に記載の人工皮膚によっても達成される。The present invention further provides (3) the content ratio of the denatured collagen is 5 to 30% by weight based on the above (1).
Alternatively, it is also achieved by the artificial skin described in (2).

【００１２】本発明はさらにまた、（４）前記アスコル
ビン酸リン酸エステルの含有比が、全体の０．１〜１０
重量％である上記（１）または（２）に記載の人工皮膚
によっても達成される。The present invention further provides (4) the content ratio of the ascorbic acid phosphoric acid ester is 0.1 to 10 of the whole.
It is also achieved by the artificial skin described in (1) or (2) above, which is in a weight percentage.

【００１３】[0013]

【作用】本発明の実施態様に基づき詳細に説明する。The operation will be described in detail based on the embodiments of the present invention.

【００１４】図１は本発明の人工皮膚の一実施態様の微
細構造を示す拡大断面図である。図１に示すように本発
明の人工皮膚１は、創傷部に接触し得る部位が細胞侵入
性の良好なコラーゲン−変性コラ−ゲンマトリックス層
からなる創傷部接触層２、該創傷部接触層２に隣接して
なる支持層３、および水蒸気透過調節層４とアスコルビ
ン酸５とからなるものである。FIG. 1 is an enlarged sectional view showing the fine structure of one embodiment of the artificial skin of the present invention. As shown in FIG. 1, the artificial skin 1 of the present invention comprises a wound contact layer 2 comprising a collagen-modified collagen matrix layer having good cell invasion at a site capable of contacting the wound site, and the wound contact layer 2. A support layer 3 adjacent to the above, a water vapor transmission control layer 4 and ascorbic acid 5.

【００１５】ここで創傷部接触層２が細胞侵入性を有す
るものとは、該人工皮膚１が創傷部に適用された際に、
マクロファージや好中球の炎症性の細胞が湿潤し、早期
に線維芽細胞が侵入し、その結果、真皮様の結合組織が
再構築されることである。Here, the wound contact layer 2 having the cell-penetrating property means that when the artificial skin 1 is applied to a wound,
It is the infiltration of inflammatory cells in macrophages and neutrophils, and early invasion of fibroblasts, resulting in the reconstruction of dermal-like connective tissue.

【００１６】また創傷部接触層２で使用する変性コラー
ゲンは、コラーゲンを加熱処理、化学処理または物理処
理等により行うことができ、特に加熱処理による熱変性
処理が最も好ましい。コラーゲンの変性度は、ヘリック
ス構造の含量によって示される。ここでヘリックス（構
造の）含量とは、分子的にコラーゲンに特有な３重鎖ヘ
リックスの含量のことであり、変性コラーゲンは、この
ヘリックスがランダムコイル化しているために、ヘリッ
クス含量がコラーゲンの変性度に対応するものである。
このヘリックス構造の含量は円偏光二色性または赤外分
光光度計で測定することができる（P.L.Gordon,I.V.Yan
nas,et al.Macromolecules, 7 (6) 954(1974)、奈倉、
橋本ら、高分子論文集 41 (8) 473 (1984))。ここで用
いられるコラーゲンの変性度の指標、すなわち、ヘリッ
クス含量は、０〜８０％、好ましくは０〜５０％、より
好ましくは３０〜５０％である。例えば、コラーゲン溶
液を６０℃、３０分間熱処理するとヘリックス含量は約
４０％であり、１００℃、２４時間熱処理するとヘリッ
クス含量は０％となることから電気泳動によりコラーゲ
ン分子の一部が切断されていることがわかる。使用する
コラーゲンは、抗原性の発現抑制の面から抗原決定基が
酵素で除去されたアテロコラーゲンが望ましく、アテロ
コラーゲンを分散状でそのまま用いると、架橋導入を行
なっても物性が余り向上しないので、アテロコラーゲン
は３７℃で中和処理（リン酸系の緩衝液を用いる）を施
し、生体内にあるような周期性線維構造を持つ線維化ア
テロコラーゲンの形にすることがさらに好ましい。The denatured collagen used in the wound contact layer 2 can be obtained by subjecting collagen to heat treatment, chemical treatment, physical treatment or the like, and heat denaturation treatment by heat treatment is most preferable. The degree of denaturation of collagen is indicated by the content of helix structure. Here, the helix (structure) content is the content of triple-chain helices that is molecularly unique to collagen, and denatured collagen has a helix content of denatured collagen because the helix is randomly coiled. It corresponds to the degree.
The content of this helix structure can be measured by circular dichroism or an infrared spectrophotometer (PLGordon, IVYan
nas, et al. Macromolecules, 7 (6) 954 (1974), Naka,
Hashimoto et al., Polymers Collection 41 (8) 473 (1984)). The index of the degree of denaturation of collagen used here, that is, the helix content is 0 to 80%, preferably 0 to 50%, and more preferably 30 to 50%. For example, when the collagen solution is heat-treated at 60 ° C. for 30 minutes, the helix content is about 40%, and when the collagen solution is heat-treated at 100 ° C. for 24 hours, the helix content becomes 0%. Therefore, a part of the collagen molecule is cleaved by electrophoresis. I understand. The collagen used is preferably atelocollagen in which an antigenic determinant is removed by an enzyme from the aspect of suppressing the expression of antigenicity, and if atelocollagen is used as it is in a dispersed state, the physical properties are not significantly improved even if cross-linking is introduced. It is more preferable to perform a neutralization treatment (using a phosphate-based buffer solution) at 37 ° C. to form a fibrotic atelocollagen having a periodic fibrous structure as in vivo.

【００１７】本発明の人工皮膚１中の変性コラーゲンの
含有比は、全体の５〜３０重量％、より好ましくは１０
〜２０重量％である。The content ratio of denatured collagen in the artificial skin 1 of the present invention is 5 to 30% by weight of the whole, more preferably 10%.
Is about 20% by weight.

【００１８】さらに水蒸気透過調節層４を設けることに
より、適度な水蒸気透過を行ない、創面に滲出液が貯留
せず、かつ創面が湿潤に保たれた状態とし、一方滲出液
中のタンパク質成分の外部への漏出は防止され、組織の
修復に極めて好ましい環境を与えることになる。Further, by providing the water vapor permeation control layer 4, the water vapor is appropriately permeated, the exudate is not stored on the wound surface, and the wound surface is kept moist while the protein component in the exudate is kept outside. Leakage is prevented and provides a highly favorable environment for tissue repair.

【００１９】本発明に用いられるアスコルビン酸５はビ
タミンＣとも呼ばれており、分子内にエンジオール基を
持ち、そのために反応性に富み、容易に酸化されてデヒ
ドロアスコルビン酸となる。アスコルビン酸５の生理活
性は、今日なお不明瞭な点が多いが、現在最も明らかに
なっている作用としてコラーゲンの生成と維持に必要な
ことが挙げられる。しかし、アスコルビン酸５は中性溶
液中３７℃で酸素存在下では、非常に不安定であった
が、Ｌ−アスコルビン酸リン酸エステルはコラーゲン合
成のコファクター活性を持ち、また生理下でも安定であ
ることが分かってきているものである。The ascorbic acid 5 used in the present invention is also called vitamin C and has an enediol group in the molecule, and therefore has high reactivity and is easily oxidized to dehydroascorbic acid. The physiological activity of ascorbic acid 5 still has many unclear points today, but one of the most clarified actions at present is that it is necessary for the production and maintenance of collagen. However, ascorbic acid 5 was extremely unstable in the presence of oxygen at 37 ° C. in a neutral solution, but L-ascorbic acid phosphate had a cofactor activity for collagen synthesis and was stable under physiological conditions. It is known to exist.

【００２０】また本発明に用いられるアスコルビン酸５
の含有量は、全体の０．１〜１０重量％が好ましく、よ
り好ましくは０．５〜２重量％である。Ascorbic acid 5 used in the present invention
The content of is preferably 0.1 to 10% by weight, and more preferably 0.5 to 2% by weight.

【００２１】本発明において、アスコルビン酸、特に活
性持続型のあるＬ−アスコルビン酸リン酸エステルを含
有することは、自己のコラーゲン線維の産生を促進する
ことができる。In the present invention, the inclusion of ascorbic acid, particularly L-ascorbic acid phosphoric acid ester having a long-acting activity, can promote the production of self collagen fibers.

【００２２】[0022]

【実施例】実施例１ アスコルビン酸含有の線維化アテロコラーゲ
ン−変性アテロコラーゲン混合溶液の調製 アテロコラーゲン（株式会社高研製）を４℃の温度下
で、ｐＨ３．０の希塩酸に溶解して０．３〜０．４ｗ／
ｖ％に調製した。この溶液を０．８μｍおよび０．２μ
ｍの直径の空孔を持つ２種のフィルターに順次通して濾
過滅菌した後、４℃に維持しつつ撹拌しながらｐＨ７．
４のリン酸緩衝液を加え、最終濃度が０．１〜０．１５
ｗ／ｖ％アテロコラーゲン（３０ｍＭリン酸ナトリウ
ム、１００ｍＭ塩化ナトリウム）であるコラーゲン溶液
とした。次いで３７℃の恒温槽内に４時間放置し、線維
化アテロコラーゲン（以下、ＦＣと記す）溶液を調製し
た。そして該ＦＣ溶液を無菌条件下で遠心操作による濃
縮を行ない、濃度を４ｗ／ｖ％に調製した。一方、フィ
ルターを順次通過させた０．３〜０．４ｗ／ｖ％のアテ
ロコラーゲン溶液を凍結乾燥し、再び無菌の蒸留水に
６．６ｗ／ｖ％となるよう再溶解し、これを６０℃の恒
温槽内に３０分間放置して熱変性を生ぜしめ変性アテロ
コラーゲン（以下、ＨＡＣと記す）溶液とした。該ＨＡ
Ｃ溶液を３７℃の温度条件下で０．４５μｍの直径の空
孔を持つフィルターを通して濾過滅菌した後、上述の４
ｗ／ｖ％ＦＣ溶液に対し、ＨＡＣ／（ＦＣ＋ＨＡＣ）＝
０．１となるように混合し、さらにＬ−アスコルビン酸
リン酸エステル（和光純薬株式会社製）を最終濃度が１
ｍＭになるように添加し、十分撹拌して、アスコルビン
酸含有の線維化アテロコラーゲン−変性アテロコラーゲ
ン混合溶液を調製した。Examples Example 1 Ascorbic acid-containing fibrotic atelocollage
Preparation of denatured atelocollagen mixed solution Atelocollagen (manufactured by Koken Co., Ltd.) was dissolved in dilute hydrochloric acid having a pH of 3.0 at a temperature of 4 ° C. to give 0.3 to 0.4 w /
It was adjusted to v%. Add this solution to 0.8 μm and 0.2 μm
After being sterilized by filtration through two kinds of filters having pores with a diameter of m, the pH is 7.
4 phosphate buffer was added to give a final concentration of 0.1-0.15.
The collagen solution was w / v% atelocollagen (30 mM sodium phosphate, 100 mM sodium chloride). Then, it was left in a constant temperature bath at 37 ° C. for 4 hours to prepare a fibrotic atelocollagen (hereinafter referred to as FC) solution. Then, the FC solution was concentrated under aseptic conditions by centrifugation to adjust the concentration to 4 w / v%. On the other hand, the 0.3 to 0.4 w / v% atelocollagen solution that has passed through the filter is freeze-dried and redissolved again in sterile distilled water to a concentration of 6.6 w / v%. The solution was left for 30 minutes in a constant temperature bath to cause heat denaturation to obtain a denatured atelocollagen (hereinafter referred to as HAC) solution. The HA
The solution C was sterilized by filtration through a filter having pores with a diameter of 0.45 μm at a temperature of 37 ° C.
HAC / (FC + HAC) = w / v% FC solution
Mix so as to be 0.1, and further add L-ascorbic acid phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) to a final concentration of 1
The mixture was added so as to be mM and sufficiently stirred to prepare a fibrotic atelocollagen-denatured atelocollagen mixed solution containing ascorbic acid.

【００２３】実施例２ 線維化アテロコラーゲンマトリ
ックスの熱脱水架橋 実施例１と同様な方法で調製した０．３ｗ／ｖ％のＦＣ
溶液を−３０℃で急速冷却した後、凍結乾燥を行ないス
ポンジを作製した。さらにこのスポンジを０．０５Ｔｏ
ｒｒ未満の真空下で１時間保持し、さらに１１０℃に温
度を上げ、２４時間真空に保ち、その後温度を室温まで
下げ、試料を取り出し、架橋した線維化アテロコラーゲ
ンスポンジを得た。 Example 2 Fibrotic Atelocollagen Matrices
Was prepared in a manner similar to thermal dehydration crosslinking in Example 1 of box 0.3 w / v% of FC
The solution was rapidly cooled at -30 ° C, and then freeze-dried to prepare a sponge. This sponge is 0.05To
It was kept under a vacuum of less than rr for 1 hour, the temperature was further raised to 110 ° C., the vacuum was kept for 24 hours, then the temperature was lowered to room temperature, and the sample was taken out to obtain a crosslinked fibrous atelocollagen sponge.

【００２４】実施例３ 人工皮膚の作製 実施例１で調製したアスコルビン酸含有の２ｗ／ｖ％線
維化アテロコラーゲン−変性アテロコラーゲン混合溶液
をステンレスパッドに注入し、さらに実施例２で調製し
た架橋した線維化アテロコラーゲンマトリックスのスポ
ンジをゆっくりのせると、スポンジは溶液の上層部に浮
く。この状態で−３０℃に急速冷却して、十分凍結させ
た後、−４０℃で０．１Ｔｏｒｒ未満の真空下で凍結乾
燥すると、線維化アテロコラーゲン−変性アテロコラー
ゲンからなる二重構造のスポンジが得られた。次にテフ
ロン上に６７％Ｓｉｌａｓｔｉｃシリコーン接着剤型Ａ
（Ｄｏｗ Ｃｏｒｎｉｎｇ社製）のヘキサン溶液を精密
被覆用具（アプリケーター）を用いて塗布し製膜した。
塗布した直後に上記のスポンジを線維化アテロコラーゲ
ンマトリックスがシリコーン側になるようにのせ、室温
で１０分程放置した後、６０℃で少なくとも１時間オー
ブンで硬化させた。さらに１１０℃に温度を上げ、４時
間真空に保ち、その後室温まで下げ、試料を取り出して
人工皮膚を得た。 Example 3 Preparation of Artificial Skin 2 w / v% fibrotic atelocollagen-denatured atelocollagen mixed solution containing ascorbic acid prepared in Example 1 was injected into a stainless steel pad, and further crosslinked fibrosis prepared in Example 2 When the sponge of the atelocollagen matrix is slowly placed, the sponge floats on the upper layer of the solution. In this state, it is rapidly cooled to −30 ° C., sufficiently frozen, and then freeze-dried at −40 ° C. under a vacuum of less than 0.1 Torr to obtain a sponge having a double structure composed of fibrotic atelocollagen and denatured atelocollagen. It was Next, 67% Silastic silicone adhesive type A on Teflon
A hexane solution (manufactured by Dow Corning) was applied using a precision coating tool (applicator) to form a film.
Immediately after the application, the sponge was placed so that the fibrotic atelocollagen matrix was on the silicone side, left at room temperature for about 10 minutes, and then cured in an oven at 60 ° C. for at least 1 hour. The temperature was further raised to 110 ° C., the vacuum was maintained for 4 hours, then the temperature was lowered to room temperature, and the sample was taken out to obtain artificial skin.

【００２５】実施例４ 人工皮膚の作製 実施例１で調製したアスコルビン酸含有の４ｗ／ｖ％線
維化アテロコラーゲン−変性アテロコラーゲン混合溶液
をステンレスパッドに注入し、−３０℃以下に急速冷却
して十分凍結させた後、−４０℃で０．１Ｔｏｒｒ未満
の真空下で凍結乾燥させることにより、線維化アテロコ
ラーゲン−変性アテロコラーゲンのマトリックスが得ら
れた。次にテフロン平板上に６７％のメディカルグレー
ドサイラスティックスシリコーン（ダウコーニング株式
会社製、接着シリコーンタイプＡ）のヘキサン溶液を精
密被覆用具（アプリケーター）を用いて塗布して製膜
し、塗布した直後に、その湿潤層上に上記の線維化アテ
ロコラーゲン−変性アテロコラーゲンマトリックスを載
せ、室温で１０分間放置した後、６０℃で少なくとも１
時間、オーブン硬化させた。さらに０．０５Ｔｏｒｒ未
満の真空下で１時間保持し、１１０℃に温度を上げ４時
間真空に保ち、その後温度を室温まで下げることによ
り、人工皮膚を得た。 Example 4 Preparation of Artificial Skin The ascorbic acid-containing 4 w / v% fibrotic atelocollagen-denatured atelocollagen mixed solution prepared in Example 1 was poured into a stainless steel pad and rapidly cooled to -30 ° C. or below and sufficiently frozen. After that, the matrix of fibrillated atelocollagen-denatured atelocollagen was obtained by freeze-drying at −40 ° C. under a vacuum of less than 0.1 Torr. Next, a hexane solution of 67% medical grade silastic silicone (manufactured by Dow Corning, adhesive silicone type A) was applied onto a Teflon plate using a precision coating tool (applicator) to form a film, and immediately after application. Then, the above-mentioned fibrotic atelocollagen-denatured atelocollagen matrix was placed on the wet layer, left at room temperature for 10 minutes, and then at least 1 at 60 ° C.
Oven cured for hours. Further, it was kept under a vacuum of less than 0.05 Torr for 1 hour, the temperature was raised to 110 ° C. and kept under vacuum for 4 hours, and then the temperature was lowered to room temperature to obtain artificial skin.

【００２６】試験 人工皮膚のラット皮膚欠損創への移
植試験 上記実施例３および４で得られたマトリックスをラット
の背部皮膚に移植して試験した。Ｗｉｓｔａｒ−ＫＹラ
ット（２００〜４００ｇ）をネンブタール麻酔下で徐毛
し、イソジン消毒したラット背部皮膚に皮下筋膜を創面
とする２０×２０ｍｍの全創皮膚欠損創を作製し、止
血、乾燥した後、生食を含ませた検体を貼付した。シリ
コーン膜辺縁を縫合糸で１６ヶ所結紫固定した。その上
にソルフレン（テルモ株式会社製）を４枚重ね、さらに
エラスチコン等の伸縮性絆創膏で胴巻きにし、圧迫固定
した。移植後、実施例３で作製した人工皮膚では１週間
後、実施例４で作製した人工皮膚では２週間後に、上層
部のシリコーンを剥がし、線維化アテロコラーゲン−変
性アテロコラーゲン層上に自家の分層植皮片を移植し
て、さらに４週間観察した。肉眼による観察では、収縮
はあまり見られず、植皮した分層植皮片が離脱していな
かった。病理組織学的にも、人工皮膚を使用して再生さ
れた真皮様組織と生着した分層植皮片はよく密着してお
り、人工皮膚内には細胞線維が多く認められた。 Test Transfer of artificial skin to rat skin defect wound
The matrix obtained in the planting Test Example 3 above and 4 were tested transplanted into the dorsal skin of rats. After Wistar-KY rats (200-400 g) were anesthetized under Nembutal anesthesia, a 20 × 20 mm total wound skin defect wound with subcutaneous fascia on the dorsal skin of the rat that had been disinfected with isodine was prepared, and after hemostasis and drying , A sample containing saline was attached. The edges of the silicone film were fixed at 16 points with a suture and fixed in purple. Four pieces of Solfrene (made by Terumo Corp.) were stacked on it, and further wrapped with a stretchable adhesive plaster such as elasticon, and fixed by compression. After transplantation, one week after the artificial skin prepared in Example 3 and two weeks after the artificial skin prepared in Example 4, the upper layer silicone was peeled off, and a self-splitting skin graft was formed on the fibrotic atelocollagen-modified atelocollagen layer. The pieces were transplanted and observed for a further 4 weeks. Macroscopically, there was not much contraction, and the grafted split-thickness skin graft pieces were not detached. Histopathologically, the dermal-like tissue regenerated using artificial skin and the grafted split-layer graft were in close contact with each other, and many cell fibers were observed in the artificial skin.

【００２７】[0027]

【発明の効果】本発明の人工皮膚は、細胞侵入性材料に
よって形成された創傷接着層と、架橋構造を有する線維
化コラーゲンマトリックスからなる支持層と、水蒸気透
過調節層とが順次積層され、少なくともいずれかの層に
活性持続型のあるＬ−アスコルビン酸リン酸エステルを
含有している人工皮膚または線維化コラーゲンとヘリッ
クス含量が０〜８０％である変性コラーゲンとのマトリ
ックスに活性持続型のあるＬ−アスコルビン酸リン酸エ
ステルを含有している創傷接着層と水蒸気透過調節層と
が積層されている人工皮膚であり、創傷、熱創、褥瘡等
により皮膚が損傷を受けた際に、損傷面に適用され、真
皮成分の欠損部位に使用することができる。EFFECT OF THE INVENTION The artificial skin of the present invention comprises a wound adhesive layer formed of a cell-penetrating material, a support layer made of a fibrotic collagen matrix having a cross-linking structure, and a water vapor permeation control layer, which are sequentially laminated, and at least L-ascorbic acid-containing artificial skin containing L-ascorbyl phosphate having an active-sustaining type in any layer or matrix of denatured collagen having a helix content of 0 to 80% -It is an artificial skin in which a wound adhesive layer containing an ascorbic acid phosphoric acid ester and a water vapor transmission control layer are laminated, and when the skin is damaged by a wound, a heat wound, a pressure ulcer, etc. It can be applied and used in the dermal component defect site.

【００２８】さらに本発明の人工皮膚は、創傷接触面が
細胞侵入性を有するので、創面に適用された際に線維芽
細胞が早期に創傷接着層に侵入し、真皮様の結合組織を
構築するので創傷の治癒が促進され、さらに移植して一
定の期間保持した後、上層の水蒸気透過調節層を剥がし
て、自家の分層植皮片を移植しても生着することができ
る。Furthermore, in the artificial skin of the present invention, since the wound contact surface has cell invasion properties, when applied to the wound surface, fibroblasts invade the wound adhesive layer early to construct a dermis-like connective tissue. Therefore, wound healing is promoted, and after transplanting and holding for a certain period of time, the upper layer water vapor permeation control layer is peeled off, and an autologous split-thickness skin graft can be transplanted for survival.

【図面の簡単な説明】[Brief description of drawings]

【図１】本発明の人工皮膚の実施態様の微細な構造を示
す断面図である。FIG. 1 is a cross-sectional view showing a fine structure of an embodiment of the artificial skin of the present invention.

【符号の説明】[Explanation of symbols]

１…人工皮膚 ２…細胞侵入性支持層 ３…耐コラゲナーゼ性支持層 ４…水蒸気透過調節層 ５…アスコルビン酸リン酸エステル DESCRIPTION OF SYMBOLS 1 ... Artificial skin 2 ... Cell-penetrating support layer 3 ... Collagenase-resistant support layer 4 ... Water vapor permeation control layer 5 ... Ascorbic acid phosphate

Claims (4)

【特許請求の範囲】[Claims] 【請求項１】 線維化コラーゲンおよびヘリックス含量
が０〜８０％である変性コラーゲンのマトリックスから
なる創傷接触層と、架橋構造を有する線維化コラーゲン
マトリックスからなる支持層と、水蒸気透過調節層とが
順次積層されてなり、少なくともいずれか１層がアスコ
ルビン酸リン酸エステルを含んでいる人工皮膚。1. A wound contact layer comprising a matrix of denatured collagen having a fibrotic collagen content and a helix content of 0 to 80%, a support layer comprising a fibrotic collagen matrix having a crosslinked structure, and a water vapor permeation control layer. An artificial skin which is laminated and at least one of which contains ascorbic acid phosphate. 【請求項２】 線維化コラーゲンとヘリックス含量が０
〜８０％である変性コラーゲンとアスコルビン酸リン酸
エステルとからなるマトリックスの支持層とその上に積
層された水蒸気透過調節層とからなることを特徴とする
人工皮膚。2. Fibrotic collagen and helix content are 0.
An artificial skin comprising a support layer of a matrix composed of denatured collagen and phosphoric acid ester of ascorbic acid of -80%, and a water vapor permeation control layer laminated thereon. 【請求項３】 前記変性コラーゲンの含有比が、全体の
５〜３０重量％である請求項１または２に記載の人工皮
膚。3. The artificial skin according to claim 1, wherein the content ratio of the denatured collagen is 5 to 30% by weight of the whole. 【請求項４】 前記アスコルビン酸リン酸エステルの含
有比が、全体の０．１〜１０重量％である請求項１また
は２に記載の人工皮膚。4. The artificial skin according to claim 1, wherein the content ratio of the ascorbic acid phosphoric acid ester is 0.1 to 10% by weight of the whole.
JP3261753A 1991-10-09 1991-10-09 Artificial skin Pending JPH05184661A (en)

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JPH05184661A true JPH05184661A (en) 1993-07-27

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002526207A (en) * 1998-09-18 2002-08-20 イムデ ビオマテリオー Double composite collagenous material, its acquisition method and therapeutic application
JP2003525705A (en) * 2000-03-09 2003-09-02 シンタコール アーゲー Multilayer collagen matrix for tissue reconstruction
JP2006334430A (en) * 2000-12-21 2006-12-14 Ethicon Inc Reinforced tissue implant and method for manufacturing and using it
EP3690029A1 (en) 2014-11-03 2020-08-05 Modern Meadow, Inc. Reinforced engineered biomaterials and methods of manufacture thereof
EP3337923B1 (en) 2015-09-21 2020-10-28 Modern Meadow, Inc. Fiber reinforced tissue composites
US11214844B2 (en) 2017-11-13 2022-01-04 Modern Meadow, Inc. Biofabricated leather articles having zonal properties
US11286354B2 (en) 2016-02-15 2022-03-29 Modern Meadow, Inc. Method for making a biofabricated material containing collagen fibrils
CN114591419A (en) * 2022-03-07 2022-06-07 山东大学 Thermal hydrolysis protein and preparation method and application thereof
US11352497B2 (en) 2019-01-17 2022-06-07 Modern Meadow, Inc. Layered collagen materials and methods of making the same

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002526207A (en) * 1998-09-18 2002-08-20 イムデ ビオマテリオー Double composite collagenous material, its acquisition method and therapeutic application
JP2003525705A (en) * 2000-03-09 2003-09-02 シンタコール アーゲー Multilayer collagen matrix for tissue reconstruction
JP2006334430A (en) * 2000-12-21 2006-12-14 Ethicon Inc Reinforced tissue implant and method for manufacturing and using it
EP3690029A1 (en) 2014-11-03 2020-08-05 Modern Meadow, Inc. Reinforced engineered biomaterials and methods of manufacture thereof
EP3337923B2 (en) † 2015-09-21 2023-01-04 Modern Meadow, Inc. Fiber reinforced tissue composites
EP3337923B1 (en) 2015-09-21 2020-10-28 Modern Meadow, Inc. Fiber reinforced tissue composites
US11913166B2 (en) * 2015-09-21 2024-02-27 Modern Meadow, Inc. Fiber reinforced tissue composites
US11286354B2 (en) 2016-02-15 2022-03-29 Modern Meadow, Inc. Method for making a biofabricated material containing collagen fibrils
US11525042B2 (en) 2016-02-15 2022-12-13 Modern Meadow, Inc. Composite biofabricated material
US11530304B2 (en) 2016-02-15 2022-12-20 Modern Meadow, Inc. Biofabricated material containing collagen fibrils
US11542374B2 (en) 2016-02-15 2023-01-03 Modern Meadow, Inc. Composite biofabricated material
US11214844B2 (en) 2017-11-13 2022-01-04 Modern Meadow, Inc. Biofabricated leather articles having zonal properties
US11352497B2 (en) 2019-01-17 2022-06-07 Modern Meadow, Inc. Layered collagen materials and methods of making the same
CN114591419A (en) * 2022-03-07 2022-06-07 山东大学 Thermal hydrolysis protein and preparation method and application thereof

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