JPH11319068A - Base material for artificial skin and production thereof - Google Patents

Base material for artificial skin and production thereof

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Publication number
JPH11319068A
JPH11319068A JP10129048A JP12904898A JPH11319068A JP H11319068 A JPH11319068 A JP H11319068A JP 10129048 A JP10129048 A JP 10129048A JP 12904898 A JP12904898 A JP 12904898A JP H11319068 A JPH11319068 A JP H11319068A
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JP
Japan
Prior art keywords
support
sponge
extracellular matrix
substrate
base material
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
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JP10129048A
Other languages
Japanese (ja)
Inventor
Takamitsu Kuroyanagi
Akihisa Sugiyama
Hiroaki Yanagawa
章寿 杉山
博昭 柳川
能光 黒柳
Original Assignee
Menicon Co Ltd
株式会社メニコン
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 Menicon Co Ltd, 株式会社メニコン filed Critical Menicon Co Ltd
Priority to JP10129048A priority Critical patent/JPH11319068A/en
Publication of JPH11319068A publication Critical patent/JPH11319068A/en
Application status is Pending legal-status Critical

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Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60Materials for use in artificial skin

Abstract

PROBLEM TO BE SOLVED: To impart strength while facilitating handling by constituting a base material of a support and a sponge formed from an extracellular matrix component.
SOLUTION: A base material for an artificial skin consists of a support 2 and a sponge (like substance) 1 formed from an extracellular matrix component. The combined form of the sponge 1 and the support 2 is set so that the sponge 1 is pref. brought into contact with at least the single surface of the support 2 and more pref. brought into contact with both surfaces thereof. The base material is produced by placing the support 2 on a prepared extracellular matrix component soln. and further adding the soln. to the surface of the support if necessary and gelling the soln. to lyophilize the same and crosslinking the extracellular matrix component layer if necessary. Herein, as the extracellular matrix component, collagen, gelatin, hyaluroinic acid or a mixture of them are used. As the support 2, a knitted fabric, a fabric or a nonwoven fabric made of a synthetic polymer such as nylon or polyester or a natural polymer such as silk or cotton is used and, pref., the knitted fabric or the fabric is used.
COPYRIGHT: (C)1999,JPO

Description

【発明の詳細な説明】 DETAILED DESCRIPTION OF THE INVENTION

【0001】 [0001]

【発明の属する技術分野】本発明は支持体と細胞外基質成分から作製されたスポンジからなる人工皮膚用基材およびその製法に関する。 With the present invention are the support and the extracellular matrix components consisting of a sponge made from artificial skin base material and their preparation. BACKGROUND OF THE INVENTION さらに詳しくは培養皮膚用基材または創傷被覆材として用いられる前記基材およびその製法に関する。 More particularly, to the substrate and their preparation for use as a substrate or a wound dressing for cultured skin.

【0002】 [0002]

【従来の技術】アテロコラーゲンなどの細胞外基質を用いて作製したスポンジ状マトリックスを培養皮膚の基材として皮膚欠損創へ適用するなど(黒柳能光、熱傷、 Such as application to skin defects wound a sponge-like matrix produced as a substrate for cultured skin using the extracellular matrix, such as the Related Art atelocollagen (Kuroyanagi Noko, burns,
「細胞組み込み型人工皮膚」第23巻、第1号、199 "Cells embedded artificial skin" Vol. 23, No. 1, 199
7年3月、9〜27頁参照)、細胞外基質からなる培養皮膚用基材および創傷被覆材が従来から使用されている。 March 1995, pp. 9 to 27), extracellular matrix consisting of cultured skin substrate and wound dressings have been used conventionally. しかしながら、前記基材は脆弱で強度が低いため、 However, since the substrate strength is low at weak,
ピンセットなどで前記基材をつまむと自重で落下したり、創面に適用する際に欠損するなど扱いづらく、また従来行なわれている移植皮膚片の固定で使用するステープル(ホッチキス)で前記基材を止めるとくずれるため、前記基材を創面に固定することはできず、非常に使いにくいものであった。 Etc. or falls by its own weight and pinch said substrate with tweezers, the substrate with staples (staples) used in fixed, including difficult to handle, also grafted skin piece is conventional deficient in applying to the wound surface since lost and stop, it can not be fixed to the substrate in the wound was achieved, very difficult to use. また、前記基材の上にガーゼまたは被覆材をそえて皮膚欠損創面まで運び適用する試みがなされているが、前記のガーゼや被覆材はそえているだけなので、適用した基材は創面に固定されず、ずれてしまうなどの問題があった。 Further, the although it on the substrate Crispy gauze or dressing an attempt to carry applied to skin defect wound surface are made fixed, since only Crispy said gauze or dressing, the applied substrate to wound surface Sarezu, there has been a problem, such as deviated.

【0003】また、とくに前記基材を培養皮膚用基材として用いたときには、細胞培養の際に用いた血清を適用前に除く洗浄操作、凍結保存後に解凍して適用する際には凍結保存液を除く洗浄操作などを行なう必要があるが、基材が脆弱であるが故に破損することが多かった。 [0003] Particularly the base material when used as a cultured skin substrate for cleaning operation to remove before applying the serum used during cell culture, cryopreservation solution in applying and decompress after cryopreservation it is necessary to perform such as cleaning operations except, substrate was often is a fragile because damage.

【0004】 [0004]

【発明が解決しようとする課題】本発明者らはこのような状況に鑑み、前記問題点を改善すべく鋭意研究を重ねた結果、支持体と細胞外基質成分から作製されたスポンジを組み合せることにより、強度が付与され取扱いやすくなった基材が提供されることを見出し、本発明を完成するに至った。 THE INVENTION Problems to be Solved] The present inventors have view of such circumstances, the result of intensive study to improve the above problems, combine the sponges made from the support and extracellular matrix components it makes found that materials based on the strength becomes easy to handle is assigned is provided, and have completed the present invention.

【0005】 [0005]

【課題を解決するための手段】すなわち、本発明は支持体と細胞外基質成分から作製されたスポンジからなる人工皮膚用基材(請求項1)に関する。 Means for Solving the Problems That is, the present invention relates to a support and extracellular matrix components consisting of a sponge made from artificial skin base (claim 1).

【0006】また、本発明は支持体と細胞外基質成分から作製されたスポンジを組み合せることからなる請求項1記載の基材の製法(請求項6)に関する。 [0006] The present invention also relates to a process for the preparation of the support and extracellular matrix components consisting of a combination of prepared sponge claim 1, wherein the base material (claim 6).

【0007】前記基材は、好ましくは、支持体と前記スポンジが組み合された請求項1記載の基材(請求項2)、細胞外基質がコラーゲン、ゼラチン、ヒアルロン酸またはそれらの混合物である請求項1記載の基材(請求項3)、支持体が合成高分子製または天然高分子製の編物または織布、または不織布である請求項1記載の基材(請求項4)および培養皮膚用基材または創傷被覆材である請求項1記載の基材(請求項5)である。 [0007] The substrate is preferably a substrate of the support and the sponge combined claims 1, wherein (claim 2), extracellular matrix collagen, gelatin, hyaluronic acid or mixtures thereof substrate of claim 1 wherein (claim 3), the support is a synthetic polymer made or natural polymer made of knitted or woven fabric or substrate according to claim 1, wherein a nonwoven fabric (claim 4) and cultured skin, a use base material or wound dressing in which claim 1 wherein the base material (claim 5).

【0008】 [0008]

【発明の実施の形態】本発明における「細胞外基質成分」とは、主に動植物からの分離・精製手法によりえられるものであって、かつ生体内で消化(分解・吸収)されるものを意味し、具体的にはコラーゲン、ゼラチン、 By "extracellular matrix component" in DETAILED DESCRIPTION OF THE INVENTION The present invention mainly be those used e by separation and purification procedures from plants and animals, and those which are digested in vivo (decomposition and absorption) meaning and, specifically, collagen, gelatin,
ヒアルロン酸、ポリグリコール酸、コンドロイチン硫酸、アルギン酸、アガロースおよびそれらの混合物などを含む。 Hyaluronic acid, polyglycolic acid, chondroitin sulfate, alginic acid, agarose, and mixtures thereof and the like. また創傷被覆材および培養皮膚用基材では創傷面周辺の線維芽細胞の活性化を促し、創傷治癒を促進することからコラーゲンが好ましい。 Also the wound dressing and cultured skin substrate promotes activation of fibroblasts around the wound surface, it collagens to promote wound healing are preferred. とくに培養皮膚用基材では、さらに容易に細胞を接着させるためにコラーゲンまたはコラーゲンとゼラチン混合物が好ましい。 In particular, cultured skin base material, collagen or collagen and gelatin mixture is preferably in order to more easily adhere to cells. 表皮細胞、線維芽細胞の移動を促進するばあいには、ヒアルロン酸またはヒアルロン酸をコラーゲンおよび/またはコラーゲンとゼラチン混合物に混合することが好ましい。 When promoting epidermal cells, the migration of fibroblasts, it is preferable to mix hyaluronic acid or hyaluronic acid collagen and / or collagen and gelatin mixture. 前記混合物の組成と混合比は用途により適宜選択しうる。 The composition and the mixing ratio of the mixture can be appropriately selected depending on the application.

【0009】前記の分離・精製手法としては、当業者が通常用いる、動植物組織から酵素処理して抽出するなどの手法があげられる。 [0009] The separation and purification techniques, used by those skilled in the art usually techniques such as extraction and enzyme treatment from animal and plant tissue.

【0010】本発明における「細胞外基質成分から作製されたスポンジ」とは、前記細胞外基質成分から作製し、スポンジ状の多孔構造を有するものであればどんなものでもよい。 [0010] "extracellular matrix sponge component made from" in the present invention, prepared from the extracellular matrix components, may be any of those having a sponge-like porous structure. 培養皮膚用基材としては、細胞ができるだけ均一にスポンジ全体に浸透し、接着できるようにするためには、空孔の径が10〜500μmであることが好ましく、30〜300μm程度であることがより好ましい。 The cultured skin base material, it to cells as uniformly as possible penetrate the entire sponge to allow the adhesive is preferably the diameter of the holes is 10 to 500 [mu] m, is about 30~300μm more preferable.

【0011】創傷被覆材としては、創面からの浸出液を充分に吸収し留置できる空孔を有することが望ましく、 [0011] The wound dressing desirably has pores that can sufficiently absorb indwelling exudate from the wound,
そのためには、空孔の径が10μm〜3mmであることが好ましく、50〜1000μm程度であることがより好ましい。 For this purpose, preferably the diameter of the pores is 10Myuemu~3mm, and more preferably about 50 to 1000 [mu] m. 空孔の径が3mmを超えると基材が脆弱となり、基材の分解も急速に起こりうるので、強度と分解速度との関係で空孔の大きさを調整する。 Diameter of pores becomes greater than 3mm the substrate is fragile, because the decomposition of the substrate may also occur rapidly, to adjust the size of the pores in relation to the strength and degradation rate.

【0012】前記スポンジの作製方法としては、たとえば、前記細胞外基質成分を適当な溶媒(たとえば水)に溶解し、ゲル化したのちに凍結乾燥し、要すれば架橋を行なう方法、適当な溶媒(たとえば水)に架橋剤を加え、成型したのちに、洗浄乾燥する方法などがあげられる。 [0012] As a manufacturing method of the sponge, for example, a method of dissolving the extracellular matrix components in a suitable solvent (e.g. water), and lyophilized After gelation, perform crosslinking, if necessary, a suitable solvent the crosslinking agent was added to (e.g., water), after which the molding and a method of washing and drying and the like.

【0013】以下に細胞外基質成分から作製されたスポンジがコラーゲンスポンジであるばあいについて、作製手順を具体的に説明する。 [0013] sponge made from extracellular matrix components below for the case where a collagen sponge, specifically illustrating a manufacturing procedure.

【0014】コラーゲンスポンジは、酸可溶性コラーゲンを用いるばあい、酸性に調製したコラーゲン溶液をホモジナイザーを用いてホモジナイズすることにより充分に気泡を含ませたものを容器に流し込み、アンモニアガス雰囲気中に静置してゲル化させたのち凍結乾燥を行ない、ついで紫外線照射または架橋剤によって分子間架橋を導入することにより作製することができる。 [0014] Collagen sponges, when using acid-soluble collagen, pouring that sufficiently moistened with bubbles by homogenizing with a homogenizer collagen solution prepared acidified to container, allowed to stand in an ammonia gas atmosphere was subjected to freeze-drying mixture was allowed to gel by, then it can be produced by introducing intermolecular cross-linked by UV irradiation or cross-linking agents.

【0015】前記コラーゲン溶液は、ウシ真皮などからえられたコラーゲンから調製して、pHを好ましくは2 [0015] The collagen solution is prepared from Karae is collagen such bovine dermis, preferably a pH 2
〜4に調整し、濃度が0.2〜3w/v%、好ましくは0.5〜2w/v%とすることによりえられる。 Was adjusted to ~ 4, concentration 0.2~3w / v%, preferably is example by a 0.5~2w / v%. ゲル化はアンモニアなどのガス雰囲気下で必要に応じて数分〜 Gelation a few minutes if necessary in a gas atmosphere such as ammonia ~
2時間程度行ない、水洗し、こののち、凍結乾燥を行なう。 Conducted about 2 hours, washed with water, Thereafter, perform lyophilization.

【0016】架橋に用いる紫外線(UV)の主波長は2 [0016] The main wavelength of ultraviolet (UV) to be used for cross-linking 2
50〜270nmのものが好ましく、紫外線量は500 Is preferably a 50~270Nm, UV dose is 500
〜12000mWsec/cm 2 、好ましくは1000 ~12000mWsec / cm 2, preferably 1000
〜5000mWsec/cm 2の線量を照射するとよい。 It is irradiated with a dose of ~5000mWsec / cm 2. 本発明に用いられる架橋剤の例としてはたとえば、 Examples of the crosslinking agent used in the present invention, for example,
グルタルアルデヒド、エチレングリコールジグリシジルエーテル、ポリグリセロールポリグリシジルエーテル、 Glutaraldehyde, ethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether,
グリセロールポリグリシジルエーテル、ヘキサメチレンジイソシアネートなどがあげられる。 Glycerol polyglycidyl ether, such as hexamethylene diisocyanate, and the like.

【0017】前記スポンジの厚さは、用途により適宜選択すればよいが、熱傷などによる皮膚欠損に適用するばあいには、表皮層および真皮層に至る欠損創を被覆する必要があるため、好ましくは0.5〜3mm、より好ましくは1〜2mm、深い褥瘡に適用するばあいには好ましくは1〜30mmである。 The thickness of the sponge, may be selected as appropriate depending on applications, because when applied to a by skin defect burns, it is necessary to cover the defect wounds lead to epidermal and dermal layers, preferably it is 0.5 to 3 mm, if more preferably to be applied 1 to 2 mm, deep decubitus preferably 1 to 30 mm.

【0018】前記スポンジの形状および大きさは適宜選択すればよいが薄板状、板状、棒状、紡錘状、両側凸レンズ状、球状などがあげられる。 [0018] The sponge shape and size may but lamellar be appropriately selected, plate-like, rod-like, spindle-shaped, each side convex lens, such as spherical and the like.

【0019】本発明における「支持体」とは、生体内で消化されない(非吸収性)構造物を意味し、たとえば、 [0019] and the "support" in the present invention means a not digested in vivo (non-absorbent) construction, for example,
ナイロン、ポリエステルまたはシリコンなどの合成高分子、絹、木綿または麻などの天然高分子からできている編物または織布、または不織布などが包含されるが、好ましくは前記編物または織布である。 Nylon, synthetic polymers such as polyester or silicone, silk, although knitted or woven fabric made from natural polymers such as cotton or hemp or nonwoven such as are encompassed, preferably the knitted or woven fabric. 前記編物または織布とは、当該人工皮膚用基材の強度を増して、基材が消化される状態においても組織と癒着しにくい程度の目を持つ編物または織布を意味するが、前記基準を満たす編物または織布、または不織布であれば、本発明に用いることができる。 Wherein A knitted or woven, increasing the strength of the artificial skin base, but the base material means a knitted or woven fabric having an eye to the extent that hardly adhesions with tissue even when being digested, the reference if knitted or woven or non-woven, meet, it can be used in the present invention. このことから、メッシュの大きさとしては、3 1 / 2 〜400メッシュでよい。 Therefore, the mesh size may be 3 1/2-400 mesh. 好ましくは、細胞を通過させ難い200〜400メッシュ、さらに好ましくは、体液を通過、蒸散させ難く創面に体液を温存させうる閉鎖包帯法(オクルーシブ)の状態に近い250〜 Preferably, the cells 200 to 400 mesh is difficult to pass through, even more preferably, it passes through the fluid, closer to the state of the occlusive dressing method which are capable of preserving the body fluid to difficult wound surface to evaporate (Okurushibu) 250
400メッシュである。 400 is a mesh. 前記メッシュとは、Tyler The mesh and is, Tyler
(テイラー)により定義されたものを意味する。 It means one defined by (Taylor).

【0020】前記不織布とは短繊維とからみ合わせ、層化させた布状物を意味し、たとえば、和紙のような形態のものをいう。 [0020] The nonwoven fabric is entangled with the short fibers, means a fabric-like material obtained by layering, for example, it refers to those forms, such as Japanese paper.

【0021】前記支持体の厚みは、用途により適宜選択すればよいが、充分に基材を支持する強度を与え、取扱いやすくするためには、好ましくは0.01〜1mmである。 The thickness of the support may be appropriately selected depending on the application, sufficiently provide strength for supporting the substrate, in order to facilitate handling, preferably 0.01 to 1 mm.

【0022】本発明の基材とは、支持体と細胞外基質成分から作製されたスポンジ(状物質)からなるものを意味する。 [0022] The base material of the present invention is one which consists of making sponge (matter) from the support and extracellular matrix components. 前記スポンジと前記支持体との組み合せの形態は、好ましくは前記支持体の少なくとも片面で前記スポンジが接触している形態であり、適用した際支持体が動かず、皮膚欠損創面へのせ、その上にガーゼをのせてとめたばあい、上方(前記創面からより遠方)のスポンジがクッションのような役割を果すので、より好ましくは前記支持体の両面に前記スポンジが接触している形態(サンドイッチ状の形態)である(図1および2参照)。 Combination of the form of the support and the sponge is in the form preferably in contact at least one surface with the sponge of the support, without moving the support when applying member, placed on the skin defect wound surface, on which If stopped topped with gauze, above since sponge (more distant from the wound surface) play a role as a cushion, form more preferable that the said sponge is in contact with both surfaces of the support (sandwich is of the form) (see FIGS. 1 and 2). 本発明の基材の形状および大きさは、適用するものにしたがい適宜選択すればよい。 Substrate shape and size of the present invention may suitably be selected in accordance with those applied.

【0023】本発明の基材の作製方法としては、前記のように調製した細胞外基質成分溶液上に支持体をのせ、 [0023] As a manufacturing method of the substrate of the present invention, the support placed on the prepared extracellular matrix components solution as described above,
要すればさらにその上に前記溶液を加えたのち、ゲル化し、凍結乾燥を行ない、要すれば架橋を行なう方法などをあげることができる。 After further the solution was added thereon if necessary, to gel, and lyophilized, it can be mentioned a method of performing cross-linking if necessary.

【0024】本発明にしたがった前記基材の製法は、好ましくは、細胞外基質成分からなる液体を支持体と前記支持体の少なくとも片面で接触させて凍結乾燥することによって支持体と細胞外基質成分から作製されたスポンジを組み合わせることからなり、より好ましくは、細胞外基質成分溶液上に支持体をのせてゲル化および凍結乾燥を行なうことによって支持体と細胞外基質成分から作製されたスポンジを組み合わせることからなり、とくに好ましくは、細胞外基質成分溶液上に支持体をのせ、その上に前記溶液を加えたのちに、ゲル化および凍結乾燥を行なうことによって支持体と細胞外基質成分から作製されたスポンジを組み合わせることからなる。 The preparation of the substrate according to the present invention, preferably, the support and the extracellular matrix by lyophilization in contact with at least one surface of a liquid consisting of the extracellular matrix component support and the support consists in combining the sponge made from components, more preferably, a sponge made from support and extracellular matrix components by the extracellular matrix component solution onto topped with support performing gelling and lyophilization It consists in combining, particularly preferably, place the extracellular matrix component solution on a support, after which the solution was added thereon, prepared from the support and extracellular matrix components by performing gel and freeze-dried It consists in combining the sponges. 前記細胞外基質成分溶液とは、細胞外基質成分と、前記細胞外基質成分を溶解する溶媒からなり、好ましくは、細胞外基質成分水溶液を意味する。 Wherein the extracellular matrix component solution, and extracellular matrix components, consists solvent for dissolving the extracellular matrix components, preferably refers extracellular matrix component solution.

【0025】本発明の基材は人工皮膚以外のいかなる用途にも用いられうるが、人工皮膚用、好ましくは培養皮膚用基材または創傷被覆材として用いられる。 The substrate of the present invention is can also be used in any application other than artificial skin, artificial skin, preferably used as a substrate or a wound dressing for cultured skin.

【0026】以下に本発明を実施例をあげてさらに詳細に説明するが、本発明はもとよりこの実施例に限定されるものではない。 [0026] The present invention by way of example will be described in more detail below, but the present invention is not intended to be limited as well in this embodiment.

【0027】 [0027]

【実施例】比較例1 コラーゲンスポンジの作製(従来技術) 1gの粉末コラーゲン((株)高研製)をpH3の塩酸酸性水溶液100mlに溶解した。 EXAMPLES dissolved preparation of Comparative Example 1 Collagen sponge (prior art) 1 g of powder collagen (Co. KoKensei) to pH3 hydrochloric acid aqueous solution 100 ml. えられたコラーゲン溶液60mlをホモジナイザー((株)日本精機製作所製)で1分間ホモジナイズして充分に気泡を含ませたのち、19cm×10cm、高さ2.5cmのポリスチレン製の容器(商品名:スチロール角型ケースNO.1 After sufficiently impregnated with bubbles The obtained collagen solution 60ml and homogenized for 1 minute in a homogenizer (Co. Nippon Seiki Seisakusho), 19cm × 10 cm, polystyrene container height 2.5 cm (trade name: styrene angle-shaped case NO.1
5、(株)サンプラテック製)に入れ、水平を維持してアンモニアガスの雰囲気下にて中和し(25℃、120 5, placed in Co. Sanplatec), while maintaining the horizontal and neutralized in an atmosphere of ammonia gas (25 ° C., 120
分)ゲル化させた。 Minute) was allowed to gel. えられたコラーゲンゲルを約−80 The obtained collagen gel about -80
℃で凍結したのち、真空凍結乾燥機(ラブコンコ社製) ℃ After freezing, the vacuum freeze dryer (Rabukonko Co.)
に移して−30℃より段階的に室温まで昇温し、30時間かけて凍結乾燥しコラーゲンスポンジをえた。 Stepwise raised to room temperature from -30 ° C. and transferred to a to give a lyophilized collagen sponge over 30 hours. えられたコラーゲンスポンジに紫外線(3600mW/cm 2 Ultraviolet gills collagen sponges (3600mW / cm 2
UV、15分)を表裏に照射して架橋した。 Crosslinked by irradiation with UV, 15 minutes) on the front and back. えられた架橋コラーゲンスポンジの形状は約18×9.5×0.1 The shape of the obtained crosslinked collagen sponge about 18 × 9.5 × 0.1
8cmであった。 It was 8cm.

【0028】また、ダルベッコ変法イーグル最小必須培地(ギブコ社製)溶液50mlを前記架橋コラーゲンスポンジに加えたばあいの前記スポンジの引裂き強度は、 Further, tear strength of the sponge when added Dulbecco's Modified Eagle's minimum essential medium (Gibco) solution 50ml to said cross-linked collagen sponge,
約0.2ニュートン/mmであり、取扱いはガーゼを添えて扱わなければならず非常に脆弱で取扱いが困難であった。 Is about 0.2 newtons / mm, handling handling was difficult in a very vulnerable must be addressed along with a gauze. なお、引裂き強度はインストロン万能材料試験機(モデル4301、インストロン社製)を用いて、2× Incidentally, tear strength using an Instron universal testing machine (Model 4301, manufactured by Instron Co.), 2 ×
3cmの長方形試験片の短辺中央にて長辺に平行に2c 3cm of at short side central rectangular test piece parallel to 2c to the long side
mの長さの切り込みを入れ、この両端を引張ることで求めた。 Cuts put a length of m, was determined by pulling the ends.

【0029】実施例1 コラーゲンスポンジと支持体からなる基材の作製 1gの粉末コラーゲン((株)高研製)をpH3の塩酸酸性水溶液100mlに溶解した。 [0029] The powder was dissolved collagen Preparation 1g of the base material made from Example 1 collagen sponge and the support (Co. KoKensei) to pH3 hydrochloric acid aqueous solution 100 ml. えられたコラーゲン溶液の60mlをホモジナイザー((株)日本精機製作所製)で1分間ホモジナイズして充分に気泡を含ませたのち、このコラーゲン溶液の30mlを19cm×10 After sufficiently impregnated with bubbles 60ml of the obtained collagen solution was homogenized for 1 minute in a homogenizer (Co. Nippon Seiki Seisakusho), 19cm × 10 to 30ml of the collagen solution
cm、高さ2.5cmのポリスチレン製の容器(商品名:スチロール角型ケースNO.15、(株)サンプラテック製)に入れ、水平を維持し、前記容器と同形状の300メッシュのナイロン製織布を前記コラーゲン溶液の上に静かにのせた。 cm, polystyrene container height 2.5 cm (trade name: Styrene Rectangular case NO.15, (Ltd.) Sanplatec) placed in, maintaining the horizontal, nylon weaving of 300 mesh of said container having the same shape the cloth was gently placed on top of the collagen solution. さらに残りのコラーゲン溶液30 Furthermore, the rest of the collagen solution 30
mlを前記ナイロン製織布の上に静かに注入した。 The ml was gently poured onto the nylon fabric. コラーゲン溶液+ナイロン製織布+コラーゲン溶液が入った容器の水平を保ち、アンモニアガスの雰囲気下で中和し(25℃、120分)ゲル化させた。 Maintaining the horizontal vessel containing a collagen solution + nylon fabric + collagen solution, neutralized (25 ° C., 120 min) in an atmosphere of ammonia gas was gels. えられたコラーゲンゲルについて比較例1と同様に凍結乾燥を行ない、コラーゲンスポンジをえた。 For the obtained collagen gel and lyophilized in the same manner as in Comparative Example 1, to give a collagen sponge. えられたコラーゲンスポンジに紫外線(3600mW/cm 2 UV、15分)を表裏に照射し架橋してえられた架橋コラーゲンスポンジの形状は約18×9.5×0.19cmであった(図3 The obtained collagen sponge ultraviolet (3600mW / cm 2 UV, 15 min) was irradiated on the front and back cross-linked form of cross-linked collagen sponge is E and was about 18 × 9.5 × 0.19cm (Figure 3
(a)〜(d)および図4(a)〜(d)参照)。 (A) ~ (d) and FIG. 4 (a) ~ (d) refer).

【0030】また、ダルベッコ変法イーグル最小必須培地(ギブコ製)溶液50mlを前記架橋コラーゲンスポンジに加えたばあいの前記スポンジは引裂き強度試験が困難なほどの強度を有しており、ガーゼなどを添えることなく、そのまま困難なく取扱うことができた。 Further, Dulbecco's Modified Eagle's minimal essential medium (Gibco) said when the solution 50ml was added to the crosslinked collagen sponge sponge has a strength enough tear strength test is difficult, add a gauze without, it could be handled without as it is difficult. なお、 It should be noted that,
引裂き強度試験は比較例1と同様に行なった。 Tear strength test was conducted in the same manner as in Comparative Example 1.

【0031】試験例1 実施例1と同様にしてえられた基材の一方のスポンジ上に10%牛胎児血清(以下、FBS)含有ダルベッコ変法イーグル最小必須培地(以下、DMEM+10%FB [0031] Test Example 1 Example 1 and one of 10% bovine fetal serum on a sponge to to E obtained substrate similar (hereinafter, FBS) containing Dulbecco's modified Eagle's minimum essential medium (hereinafter, DMEM + 10% FB
S、ギブコ社製)中に懸濁した培養ウサギ線維芽細胞を1×10 4細胞/cm 2の密度で播種したのちCO 2 S, CO 2 5 After seeded at a density of cultured rabbit fibroblasts 1 × 10 4 cells / cm 2 suspended in Gibco) in
%、35℃のインキュベーター中で7日間培養し、培養真皮を作製した。 %, And cultured in a 35 ° C. incubator for 7 days, to prepare a culture dermis. 培地を除去し、これを凍結保存液(1 The medium was removed and stored frozen solution which (1
0%グリセロール含有DMEM+10%FBS)に入れ−152℃の冷凍庫内で保存した。 Was stored in a freezer at 0% glycerol containing DMEM + 10% FBS) put -152 ° C.. 3カ月間凍結保存したのち、解凍して凍結保存液を除去し、ハンクス液30 3 months After months frozen storage, was removed and stored frozen solution was thawed, Hank's solution 30
mlで2回洗浄して動物実験に使用した。 It was washed twice was used in the animal experiments ml. ウサギの背部に直径7cmの円を描き前層皮膚を切除した。 It was excised before layer of skin on the back of a rabbit draw a circle with a diameter of 7cm. 切除後、 After resection,
皮膚欠損創の直径は9cmと広がった。 The diameter of the skin defect wounds spread and 9cm. この皮膚欠損創に解凍した前記基材(同種培養真皮)を直径9cmに切って適用し創周辺をナイロン6.0縫合糸(協和時計工業(株)製)により縫合し、その上にバイオクルーシブ(ポリウレタンフィルム製創傷被覆材)を適用し、さらに直径9cmの滅菌パットをのせて、前記と同様に創周辺と縫合固定し伸縮性包帯で圧迫固定した。 Sutured This skin defects wounds the substrate thawed nylon 6.0 suture applying surrounding wound cut into (allogeneic cultured dermis) diameter 9cm (Kyowa Clock Industry Co.), Bio crew thereon apply the Sibu (polyurethane film made dressings), further topped with sterile pad with a diameter of 9cm, was compressed and secured in the same manner as described above wound around the suture fixed elastic bandage. 1週間後に、包帯交換を行ない創面を観察して、再度、新しい基材を適用し、さらに2週間(3週目)に創面を観察した。 After one week, to observe the wound surface subjected to dressing changes, again, to apply the new substrate, it was observed wound surface further 2 weeks (3 weeks).

【0032】コラーゲンスポンジ内にはナイロンメッシュが組み込まれているため解凍後の洗浄操作は非常に容易であり、また移植操作が容易であった。 The washing operation after thawing for the in collagen sponges has been incorporated nylon mesh is very easy, transplantation operation was easy. とくに創周辺との縫合固定が可能であった。 In particular, it was possible to suture fixation of the peripheral wounds. 3週目において良好な肉芽組織が形成され、創周辺からの表皮化も観察された。 In the third week is good granulation tissue formation was also observed epidermalization from around wounds.
新生組織はナイロンメッシュに食い込んでおらず容易に創面からナイロンメッシュを除去することができた。 Neoplastic tissue was able to remove the nylon mesh from easily wound surface does not bite into the nylon mesh.

【0033】 [0033]

【発明の効果】本発明によれば、支持体と細胞外基質成分から作製されたスポンジを組み合せることにより、これらからなる基材の強度が向上され取扱いやすくなった人工皮膚用基材を提供することが可能となる。 According to the present invention, provides support and extracellular By combining fabricated sponge from matrix components, handled easily since artificial skin base material strength of the substrate made of these is improved it is possible to become. さらに、 further,
本発明の基材のスポンジ上に細胞を播種することにより培養皮膚が大量に生産でき、凍結保存が可能で、要時解凍し使用できる。 Can be produced in large quantities is cultured skin by seeding the cells onto the sponge base material present invention, can be stored frozen, thawed it can be used when needed. えられた培養皮膚は解凍後の凍結保存液を除去するための洗浄操作性、輸送性および移植操作性が向上されており、また創面をオクルーシブに近い状態にできるので、創面の組織修復が促進されることになり、適当な時期に新生組織に悪影響を与えることなく創面から支持体を除去することができる。 The obtained cultured skin cleaning operability for removing the cryopreservation solution after thawing, transporting and are improved portability operability, and because possible wound surface in a state close to Okurushibu, wound surface tissue repair promoting are the results in, can be removed the support from the wound without adversely affecting the neoplastic tissue at an appropriate time. 本発明の基材は、培養皮膚用基材と同様、創傷被覆材としても好適に用いられる。 The substrate of the present invention, as well as cultured skin base material is preferably used as a wound dressing.

【図面の簡単な説明】 BRIEF DESCRIPTION OF THE DRAWINGS

【図1】本発明の基材の一実施例の説明図であって、スポンジとスポンジの間に支持体がはさまった状態で支持体とスポンジが組み合されている基材の断面図を示す図である。 [1] is an explanatory view of an example of a substrate of the present invention, shows a cross-sectional view of the support and the substrate sponge is combined in a state in which the support is sandwiched between the sponge and the sponge it is a diagram.

【図2】本発明の基材の別の一実施例の説明図であって、支持体の片面でスポンジが接触している状態で支持体とスポンジが組み合されている基材の断面を示す図である。 [Figure 2] is an explanatory view of another embodiment of a substrate of the present invention, the cross section of the support and the substrate sponge is combined in a state of sponge on one side of the support is in contact It illustrates.

【図3】本発明の基材の一作製手順(前半)を示す説明図である。 3 is an explanatory diagram showing an production procedure (first half) of the base material of the present invention.

【図4】本発明の基材の一作製手順(後半)を示す説明図である。 [4] One manufacturing procedure (second half) of the base material of the present invention is an explanatory diagram showing.

【符号の説明】 DESCRIPTION OF SYMBOLS

1 スポンジ 2 支持体 3 コラーゲン溶液 4 コラーゲンゲル 11 コラーゲンスポンジ 12 架橋コラーゲンスポンジ 21 ナイロンメッシュ 1 Sponge 2 support 3 collagen solution 4 collagen gel 11 collagen sponge 12 cross-linked collagen sponge 21 nylon mesh

Claims (6)

    【特許請求の範囲】 [The claims]
  1. 【請求項1】 支持体と細胞外基質成分から作製されたスポンジからなる人工皮膚用基材。 1. A support and extracellular matrix components consisting of a sponge made from artificial skin base.
  2. 【請求項2】 支持体と前記スポンジが組み合された請求項1記載の基材。 Wherein said the support sponge was combined claim 1 Substrate according.
  3. 【請求項3】 細胞外基質がコラーゲン、ゼラチン、ヒアルロン酸またはそれらの混合物である請求項1記載の基材。 Wherein the extracellular matrix is ​​collagen, gelatin, hyaluronic acid or base according to claim 1, wherein a mixture thereof.
  4. 【請求項4】 支持体が合成高分子製または天然高分子製の編物または織布、または不織布である請求項1記載の基材。 Wherein the support is a synthetic polymer made or natural polymer made of knitted or woven fabric or substrate according to claim 1, wherein the non-woven fabric.
  5. 【請求項5】 培養皮膚用基材または創傷被覆材である請求項1記載の基材。 5. The substrate of claim 1 wherein the cultured skin for a substrate or a wound dressing.
  6. 【請求項6】 支持体と細胞外基質成分から作製されたスポンジを組み合せることからなる請求項1記載の基材の製法。 6. The method of support and extracellular matrix components consisting of a combination of prepared sponge claim 1, wherein the substrate.
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JP2012080915A (en) * 2010-10-06 2012-04-26 Hitachi Chemical Co Ltd Wound covering material
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