JP2004194944A - Substrate for cartilage cultivation and its manufacturing method - Google Patents
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Abstract
Description
【0001】
【発明の属する技術分野】
本発明は、細胞培養によって軟骨組織を再生させる際に使用される培養用基材および、その製造法の提供に関する。
【0002】
【従来の技術】
手術、外傷、関節の変形性疾患等によって失われた軟骨組織を細胞培養によって再構築し、人体に移植することにより再生する手法が再生医療として実用化されつつある。
即ち、近年の細胞培養技術の進歩により、種々の細胞の培養が可能となり、例えば、桟敷らはコラーゲンスポンジに軟骨細胞を播種し、軟骨組織を再生させることに成功している(非特許文献1参照)。また、Vacantiらは生体内吸収性高分子であるグリコール酸と乳酸の共重合体からなる繊維を基材としてこれに軟骨細胞を播種することによって軟骨組織の再生を試みている(非特許文献2参照)。また、生体内分解性ポリマーやその多孔体を軟骨細胞の培養用基材として用いることも知られている(特許文献1、特許文献2参照)。さらに、本出願人において、乳酸、グリコール酸もしくはカプロラクトンのいずれかの重合体またはこれらの共重合体のいずれかからなり、細胞保持構造を有するスポンジ状成型物による軟骨組織再生用基材も提案している(特許文献3参照)。
【0003】
【非特許文献1】
Biomaterials 17(1996)155-162
【非特許文献2】
Plast.Reconstr.Surg.88,753,1991
【特許文献1】
WO90/2091号公報
【特許文献2】
WO98/31345号公報
【特許文献3】
特開平10−234844号公報
【0004】
【発明が解決しようとする課題】
然しながら、上記構成によるものは、取扱い性、任意形状・大きさへの切断の容易性、縫合性、細胞の増殖効率等においてなお改善の余地があった。
【0005】
【課題を解決するための手段】
本発明は上記課題を解決するために成されたもので、以下の構成に特徴を有する。
項1.架橋されたコラーゲンスポンジを構成素材としたことを特徴とする軟骨培養用基材。
項2.1ミクロン〜500ミクロンのポアサイズを有するコラーゲンスポンジである項1記載の軟骨培養用基材。
項3.厚さが1〜20mmの範囲にある項1〜2に記載の軟骨培養用基材。
項4.生体内分解吸収性の強化材と複合化されていることを特徴とする項1〜3に記載の軟骨培養用基材。
項5.強化材が片面もしくは中心部に位置して一体化されている項4に記載の軟骨培養用基材。
項6.強化材が網目状に構成されたものである項4に記載の軟骨培養用基材。
項7.強化材が編、織地、不織布、フィルムから選択されたものである項6に記載の軟骨培養用基材。
項8.強化材が乳酸、グリコール酸、カプロラクトン、の重合体およびこれらの共重合体、もしくは、重合体の混合物より選択されたものである項7に記載の軟骨培養用基材。
項9.強化材が親水化処理されたものである項8に記載の軟骨培養用基材。
項10.架橋されたコラーゲンスポンジと強化材が生体内分解吸収性ポリマーによって接着されている項4に記載の軟骨培養用基材。
項11.接着に用いられる生体内分解吸収性ポリマーが乳酸とカプロラクトンの共重合体である項10に記載の軟骨培養用基材。
項12.強化材を敷設した上からコラーゲンの水溶液、または、酢酸、もしくは、エタノールとの混合溶液、あるいは、これらの発泡液を注ぎ、次いで凍結乾燥することを特徴とする軟骨培養用基材の製造法。
項13.強化材を敷設した上から生体内分解吸収性ポリマー溶液を注ぎ、コラーゲンスポンジをのせ、凍結乾燥することにより、強化材とコラーゲンスポンジとを接着させることを特徴とする軟骨培養用基材の製造法。
【0006】
【発明の実施の形態】
本発明は特に、多孔体足場(スポンジ)として適度に架橋されたコラーゲンを使用し、生体内分解性素材による強化材により補強されていることに特徴を有し、これにより、取扱い性の向上、任意形状・大きさへの切断の容易性、縫合性、細胞の増殖効率等の改善がなされたものである。
以下、かかる構成について詳細に説明する。
【0007】
(架橋コラーゲンスポンジ)
本発明に使用されるコラーゲンは、皮膚、骨、腱、或いは、動物、ヒト等の由来を問わず、変性コラーゲン、線維化コラーゲン等、その構造に関わらず目的を達することができるが、特に、抗原性を低下させる目的で末端のテロぺプタイドを酵素的に除去したアテロコラーゲンの使用が好ましい。
また、架橋処理は生体内での分解のコントロールと培養液につけた際、膨潤しすぎて取扱い難くなる欠点を解消することを主目的として行う。その方法としては、熱架橋、紫外線架橋、或いは、グルタルアルデヒド、ヘキサメチレンジイソシアネート、カルボジイミド等を用いた化学架橋を用いることができる。架橋度の調整は、これらの条件を適宜選択して行えばよいが、通常5日〜数ヶ月の範囲、望ましくは、数週間〜3ヶ月程度で分解するよう調整するのが好ましい。具体的には含水率が60%〜95%の範囲への調整が目安となる。
一方、スポンジ化は、例えば、コラーゲン水溶液、または、酢酸、もしくは、エタノールとの混合溶液、あるいは、これらの発泡液を凍結乾燥することにより行う。この際、コラーゲンの水溶液にクロロホルムを加えたり、酢酸を加えたり、エタノールを加えることにより、ポーラスの安定性や均一性を向上させることができる。また、溶液の濃度、攪拌の程度、溶媒、溶媒への添加物、凍結乾燥条件を適宜変えることにより、ポアサイズを数ミクロン〜数百ミクロン、具体的には1ミクロン〜500ミクロン、厚さ1〜20mmとすることが望まれる。何れもこの範囲内であることは、軟骨細胞の増殖性において、また、強度、取扱いの利便性等において好適な範囲である。さらに、かかる構成においては、乳酸−カプロラクトン共重合体のスポンジ体と複合化、例えば、貼り合わせたり、重ねたりした構成としてもよい。
【0008】
(強化材)
生体内分解吸収性高分子であるポリ乳酸、乳酸―カプロラクトン共重合体、ポリグリコール酸、乳酸―グリコール酸共重合体、乳酸−エチレングリコール共重合体、ポリジオキサノン、グリコール酸−カプロラクトン共重合体、グリコール酸−トリメチレンカーボネート共重合体が例示できるが、本発明においては、分解性の制御、柔軟性において、乳酸、グリコール酸、カプロラクトン、の重合体およびこれらの共重合体、もしくは、重合体の混合物より選択されたものであることが好ましい。
本発明においては、これらの高分子を公知の方法により紡糸、延伸した糸を用いて編成、織成、不織布化、平組紐化等して得た布状体、或いは、押出し、キャスティング等して得たフィルムを強化材として用いる。なお、不織布化の方法としては、ニードルパンチング方式のほか、メルトブロー方式により得たものを用いることもできる。
なお、これらの強化材は、培養される細胞への栄養補給を考慮し、網目状(メッシュ状)で、特にその目付けを5〜200g/m2の範囲とすることが好ましい。従って、フィルムに関しては、適宜の方法により、微細な孔を設けることが望ましい。さらに、コラーゲンスポンジとの接着性を向上させる目的において、予め親水化処理が施されることが望ましい。
かかる親水化の方法としては、例えば、プラズマ処理、グロー放電処理、コロナ放電処理、オゾン処理、表面グラフト処理、コーティング処理、薬品処理等が挙げられるが、操作性、効果の面からプラズマによる処理が望ましい。
【0009】
(複合化の方法)
本発明においては、その複合化の形態は、コラーゲンスポンジの片面に強化材を一体化させるもの、或いは、強化材を中心にその両面をコラーゲンスポンジによりサンドイッチ状に挟んだ形態とするものが例示できる。また、強化材はその全面に配置することも、一部に配置することも任意であるが、取扱いの利便性の面より全面に配置させることが好ましい。さらに、一体化のために生体内分解吸収性ポリマー溶液を接着剤として用いてもよい。かかる接着剤としては、乳酸−カプロラクトン共重合体溶液が好ましい例として例示できる。
以下に複合化の方法について具体的に例示するが、これに限定されない。
(A法)
適宜の容器に強化材を敷く⇒ホモジナイズしたコラーゲン水溶液、または、酢酸、もしくは、エタノールを加えた水溶液を注ぐ⇒凍結乾燥する⇒架橋処理する(2層構造)
(B法)
適宜の容器に強化材を敷く⇒接着剤としての乳酸−カプロラクトン共重合体溶液を注ぐ⇒架橋したコラーゲンスポンジをその上に貼り付ける⇒凍結乾燥する(接着層を介した2層構造)
(C法)
適宜の容器にホモジナイズしたコラーゲン溶液を注ぐ⇒強化材を載せる⇒ホモジナイズしたコラーゲン水溶液、または、酢酸、もしくは、エタノールを加えた水溶液を注ぐ⇒凍結乾燥・架橋する(サンドイッチ状の3層構造)
以下、具体的に実施例を挙げて説明する。
【0010】
【実施例1】
1.コラーゲンスポンジの作製
・アテロコラーゲン(新田ゼラチン(株)製)を原料とし、3mg/ml、pH3.0のコラーゲン水溶液を調整する。
・この溶液50gにクロロホルム0.5gを添加し、ホモジナイザーを用いて、6000rpmで1分間、激しく撹拌する。
・得られたクリーム状の発泡液をアルミ製型枠(11cm×8.5cm)に流し込む。
・−40℃の冷凍庫内で1時間静置し、凍結させる。
・真空減圧下(0.01mmHg)で−40°Cから40°Cまで12時間かけて一定速度で加温し、さらに真空減圧下40°Cで12時間乾燥させコラーゲンスポンジを得る。
2.熱架橋コラーゲンスポンジの作製
・上記のコラーゲンスポンジに真空減圧下105°Cで24時間加熱乾燥を行い、熱架橋コラーゲンスポンジを得る。
3.化学架橋コラーゲンスポンジの作製
・上記の熱架橋コラーゲンスポンジを5°Cの0.2wt%のグルタルアルデヒド/酢酸溶液中に、24時間浸漬させ、化学架橋させる。
・得られたスポンジをイオン交換水で十分洗浄する。
・洗浄後、15%エタノール水溶液に浸漬させる。
・-135°Cの冷凍庫内で12時間静置し、凍結させる。
真空減圧下(0.01mmHg)で−40°Cから40°Cまで12時間かけて一定速度で加温し、さらに真空減圧下40℃で12時間乾燥させ化学架橋複合化コラーゲンスポンジを得る。
4.ポリ−L乳酸繊維(布)との複合化
・ ポリ−L乳酸繊維(PLLA繊維)から補強用布を作製する。
(組織:フライス編生地、両面編生地、ツインニット生地、平織生地、天竺編生地、不織布の7種類)
・乳酸−カプロラクトン共重合体(P(LA/CL) ;重合比50:50)の4%ジオキサン溶液を作製する。
・20cm×20cmのガラス型枠に前記のPLLA布を敷き、そこへP(LA/CL)溶液を流し込む。
・その上に前記3で得たスポンジをのせ、速やかに−80°Cの冷凍庫に移し、凍結させる。
・ 真空減圧下(0.01mmHg)で−40°Cから40°Cまで12時間かけて一定速度で加温し、さらに真空減圧下40°Cで6時間乾燥させ、さらに真空減圧下70°Cで12時間乾燥させ、コラーゲンスポンジとPLLA布およびP(LA/CL)スポンジの複合体を得る。
【0011】
【実施例2】
1.乳酸−カプロラクトン補強材(布)の調整
・乳酸−カプロラクトン共重合体(P(LA/CL);重合比75:25)繊維から布を作製する。(組織:フライス編生地、両面編生地、ツインニット生地、平織生地、天竺編生地、不織布の7種類)
・ P(LA/CL)布をプラズマ処理(酸素、100W、0.5torr、30秒)し、親水化する。2.コラーゲンスポンジとの複合化
・アテロコラーゲン(新田ゼラチン)を原料とし、3mg/ml、pH3.0のコラーゲン水溶液を調整する。
・この溶液50gにクロロホルム0.5gを添加し、ホモジナイザーを用いて、6000rpmで1分間、激しく撹拌する。
・アルミ製型枠(11cm×8.5cm)に前記親水化したP(LA/CL)布を敷き、得られたクリーム状のコラーゲン発泡液を流し込む。
・−40°Cの冷凍庫内で1時間静置し、凍結させる。
・真空減圧下(0.01mmHg)で−40°Cから40°Cまで12時間かけて一定速度で加温し、さらに真空減圧下40°Cで12時間乾燥させ、P(LA/CL)補強布と複合化したコラーゲンスポンジを得る。
3.熱架橋
上記の複合体に真空減圧下105°Cで24時間加熱乾燥を行い、熱架橋させる。
4.化学架橋
・前記の複合化物を5°Cの0.2wt%のグルタルアルデヒド/酢酸溶液中に、24時間浸漬させ、化学架橋させる。
・得られたスポンジをイオン交換水で十分洗浄する。
・洗浄後、15%エタノール水溶液に浸漬させる。
・-135°Cの冷凍庫内で12時間静置し、凍結させる。
・ 真空減圧下(0.01mmHg)で−40°Cから40°Cまで12時間かけて一定速度で加温し、さらに真空減圧下40℃で12時間乾燥させ化学架橋複合化コラーゲンスポンジを得る。
【0012】
(動物への移植)
ウサギの骨髄を採取し、15%ウシ胎児血清を含むDMEM培地と混和し、約1週間培養した。さらに約1週間継代培養し、106程度に増殖させた。この細胞を直径4mm、厚さ3mmの円柱状に切り出した上記実施例2で得たフライス編生地と架橋コラーゲンスポンジとの複合体に播種した。ウサギの膝関節を露出させ、ドリルを用いて直径4mm深さ4mmの骨軟骨欠損を作製し、細胞を播種したサンプルを欠損部に挿入した。手術後1,2,4週間でウサギを犠牲死させ、移植部位の組織切片を作製した。術後、時間の経過とともに、関節面での軟骨組織の再生および骨欠損部での骨組織の再生が認められた。
【0013】
【発明の効果】
本発明は、コラーゲンの架橋、強化材による補強効果により、取扱い利便性の向上、自由な形状、大きさへの切断が可能、十分な縫合強度が付加されること、補強材が吸収されること、補強材が多孔体(適度に目が開いている)であるので栄養透過すること等、多大な特徴、効果を有するものである。
よって、軟骨再生用としては勿論のこと、他の器官の再生用基材として、例えば、培養皮膚用基材としても用いることができる。特に、サンドイッチ構造による3層タイプのものは培養皮膚基材として好適に用いることができるものである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a culture substrate used for regenerating cartilage tissue by cell culture and a method for producing the same.
[0002]
[Prior art]
A technique of regenerating cartilage tissue lost by surgery, trauma, joint deformity, etc. by cell culture and transplanting it into the human body is being put into practical use as regenerative medicine.
That is, recent advances in cell culture technology enable the cultivation of various cells. For example, Kashiki et al. Have succeeded in seeding chondrocytes in a collagen sponge and regenerating cartilage tissue (Non-patent Document 1). reference). Vacanti et al. Attempted to regenerate cartilage tissue by seeding chondrocytes on a fiber composed of a copolymer of glycolic acid and lactic acid, which is a bioabsorbable polymer, as a base material (Non-patent Document 2). reference). It is also known to use a biodegradable polymer or a porous body thereof as a substrate for culturing chondrocytes (see Patent Document 1 and Patent Document 2). Furthermore, the present applicant has also proposed a base material for cartilage tissue regeneration using a sponge-like molded product comprising a polymer of any of lactic acid, glycolic acid or caprolactone, or a copolymer thereof, and having a cell holding structure. (See Patent Document 3).
[0003]
[Non-Patent Document 1]
Biomaterials 17 (1996) 155-162
[Non-Patent Document 2]
Plast.Reconstr.Surg.88,753,1991
[Patent Document 1]
WO90 / 2091 [Patent Document 2]
WO98 / 31345 [Patent Document 3]
Japanese Patent Laid-Open No. 10-234844
[Problems to be solved by the invention]
However, the above configuration still has room for improvement in handling, ease of cutting into an arbitrary shape and size, stitchability, cell proliferation efficiency, and the like.
[0005]
[Means for Solving the Problems]
The present invention has been made to solve the above problems, and has the following features.
Item 1. A base material for cartilage culture characterized by comprising a cross-linked collagen sponge as a constituent material.
Item 2. The substrate for cartilage culture according to Item 1, which is a collagen sponge having a pore size of Item 2.1 to 500 microns.
Item 3. Item 3. The substrate for cartilage culture according to Item 1-2, wherein the thickness is in the range of 1-20 mm.
Item 4. Item 4. The cartilage culture substrate according to any one of Items 1 to 3, which is combined with a biodegradable and absorbable reinforcing material.
Item 5. Item 5. The cartilage culture substrate according to Item 4, wherein the reinforcing material is integrated on one side or in the center.
Item 6. Item 5. The base material for cartilage culture according to Item 4, wherein the reinforcing material is configured in a mesh shape.
Item 7. Item 7. The base material for cartilage culture according to Item 6, wherein the reinforcing material is selected from knitted fabric, woven fabric, non-woven fabric, and film.
Item 8. Item 8. The cartilage culture substrate according to Item 7, wherein the reinforcing material is selected from a polymer of lactic acid, glycolic acid, caprolactone, a copolymer thereof, or a mixture of polymers.
Item 9. Item 9. The cartilage culture substrate according to Item 8, wherein the reinforcing material has been subjected to a hydrophilic treatment.
Item 10. Item 5. The cartilage culture substrate according to Item 4, wherein the cross-linked collagen sponge and the reinforcing material are bonded together with a biodegradable absorbable polymer.
Item 11. Item 11. The substrate for cartilage culture according to Item 10, wherein the biodegradable absorbable polymer used for adhesion is a copolymer of lactic acid and caprolactone.
Item 12. A method for producing a substrate for cartilage culture, which comprises pouring an aqueous solution of collagen, a mixed solution with acetic acid or ethanol, or a foamed solution thereof from the laid reinforcing material, and then freeze-drying.
Item 13. A method for producing a substrate for cartilage culture, comprising pouring a biodegradable absorbable polymer solution from a reinforcing material, placing a collagen sponge, and lyophilizing to adhere the reinforcing material and the collagen sponge. .
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is particularly characterized in that it uses moderately cross-linked collagen as a porous scaffold (sponge) and is reinforced by a reinforcing material made of a biodegradable material, thereby improving handling properties, Improvements such as ease of cutting into arbitrary shapes and sizes, stitchability, and cell growth efficiency have been made.
Hereinafter, this configuration will be described in detail.
[0007]
(Cross-linked collagen sponge)
Collagen used in the present invention can achieve the purpose regardless of its structure, whether it is derived from skin, bone, tendon, animal, human or the like, denatured collagen, fibrotic collagen, etc. For the purpose of reducing antigenicity, it is preferable to use atelocollagen in which the terminal telopeptide is enzymatically removed.
The main purpose of the cross-linking treatment is to eliminate the disadvantage that it becomes difficult to handle due to excessive swelling when it is attached to a culture solution and control of in vivo degradation. As the method, thermal crosslinking, ultraviolet crosslinking, or chemical crosslinking using glutaraldehyde, hexamethylene diisocyanate, carbodiimide, or the like can be used. The degree of cross-linking may be adjusted by appropriately selecting these conditions. However, it is preferable that the degree of crosslinking is usually adjusted in the range of 5 days to several months, preferably in the range of several weeks to 3 months. Specifically, adjustment to a moisture content range of 60% to 95% is a standard.
On the other hand, sponge formation is performed, for example, by freeze-drying a collagen aqueous solution, a mixed solution with acetic acid or ethanol, or a foamed solution thereof. At this time, the stability and uniformity of the porous material can be improved by adding chloroform to the collagen aqueous solution, adding acetic acid, or adding ethanol. Further, by appropriately changing the concentration of the solution, the degree of stirring, the solvent, the additive to the solvent, and the lyophilization conditions, the pore size is several microns to several hundred microns, specifically 1 micron to 500 microns, and the thickness is 1 to It is desired to be 20 mm. The fact that both are within this range is a preferable range in terms of chondrocyte proliferation, strength, convenience of handling, and the like. Furthermore, in this structure, it is good also as a structure which compounded with the sponge body of the lactic acid-caprolactone copolymer, for example, bonded together or piled up.
[0008]
(Reinforcing material)
Biolactic acid, lactic acid-caprolactone copolymer, polyglycolic acid, lactic acid-glycolic acid copolymer, lactic acid-ethylene glycol copolymer, polydioxanone, glycolic acid-caprolactone copolymer, glycol An acid-trimethylene carbonate copolymer can be exemplified, but in the present invention, in terms of control of degradability and flexibility, a polymer of lactic acid, glycolic acid, caprolactone and a copolymer thereof, or a mixture of these polymers It is preferable that it is more selected.
In the present invention, these polymers are spun and stretched by a known method using a knitted, woven, non-woven fabric, flat braid, etc., or extruded, cast, etc. The obtained film is used as a reinforcing material. In addition, as a method for forming the nonwoven fabric, in addition to the needle punching method, those obtained by the melt blow method can also be used.
These reinforcing materials are in the form of a mesh (mesh shape) in consideration of the nutritional supply to the cells to be cultured, and the basis weight is particularly preferably in the range of 5 to 200 g / m 2 . Therefore, regarding the film, it is desirable to provide fine holes by an appropriate method. Furthermore, it is desirable to perform a hydrophilic treatment in advance for the purpose of improving the adhesion with the collagen sponge.
Examples of such hydrophilic methods include plasma treatment, glow discharge treatment, corona discharge treatment, ozone treatment, surface graft treatment, coating treatment, chemical treatment, etc., but treatment with plasma is preferable from the viewpoint of operability and effects. desirable.
[0009]
(Method of compounding)
In the present invention, examples of the composite form include a form in which a reinforcing material is integrated on one side of a collagen sponge, or a form in which both sides of a reinforcing material are sandwiched between collagen sponges. . Further, the reinforcing material may be arranged on the entire surface or a part thereof, but it is preferable to arrange the reinforcing material on the entire surface from the viewpoint of convenience of handling. Furthermore, a biodegradable absorbable polymer solution may be used as an adhesive for integration. An example of such an adhesive is a lactic acid-caprolactone copolymer solution.
The compounding method is specifically illustrated below, but is not limited thereto.
(Method A)
Put a reinforcing material in an appropriate container ⇒ pour a homogenized collagen aqueous solution or an aqueous solution containing acetic acid or ethanol ⇒ freeze-dry ⇒ cross-link treatment (two-layer structure)
(Method B)
Put a reinforcing material in an appropriate container ⇒ pour a lactic acid-caprolactone copolymer solution as an adhesive ⇒ stick a cross-linked collagen sponge on it ⇒ freeze-dry (two-layer structure with an adhesive layer)
(Method C)
Pour the homogenized collagen solution into an appropriate container ⇒ Place the reinforcing material ⇒ Pour the homogenized collagen aqueous solution or the aqueous solution with acetic acid or ethanol added ⇒ Freeze-dry and cross-link (sandwich three-layer structure)
Hereinafter, specific examples will be described.
[0010]
[Example 1]
1. Preparation of collagen sponge-Using atelocollagen (manufactured by Nitta Gelatin Co., Ltd.) as a raw material, a 3 mg / ml, pH 3.0 collagen aqueous solution is prepared.
Add 0.5 g of chloroform to 50 g of this solution, and vigorously stir for 1 minute at 6000 rpm using a homogenizer.
-Pour the resulting creamy foam into an aluminum mold (11cm x 8.5cm).
・ Leave for 1 hour in a -40 ℃ freezer and freeze.
Heat at a constant rate over 12 hours from −40 ° C. to 40 ° C. under vacuum reduced pressure (0.01 mmHg), and further dry at 40 ° C. under vacuum reduced pressure for 12 hours to obtain a collagen sponge.
2. Preparation of heat-crosslinked collagen sponge • The above-described collagen sponge is heat-dried at 105 ° C under vacuum for 24 hours to obtain a heat-crosslinked collagen sponge.
3. Preparation of Chemically Crosslinked Collagen Sponge • The above-mentioned thermally cross-linked collagen sponge is immersed in a 0.2 wt% glutaraldehyde / acetic acid solution at 5 ° C. for 24 hours for chemical crosslinking.
-Wash the resulting sponge thoroughly with ion-exchanged water.
-After washing, immerse in 15% ethanol aqueous solution.
・ Leave in a freezer at -135 ° C for 12 hours to freeze.
Heat at a constant rate from −40 ° C. to 40 ° C. under vacuum under reduced pressure (0.01 mmHg) over 12 hours, and further dry at 40 ° C. under vacuum under reduced pressure for 12 hours to obtain a chemically crosslinked complexed collagen sponge.
4). Composite with poly-L lactic acid fiber (cloth)-A reinforcing cloth is prepared from poly-L lactic acid fiber (PLLA fiber).
(Organization: 7 types: milling fabric, double-sided knitting fabric, twin knitted fabric, plain woven fabric, tengu knitted fabric, non-woven fabric)
-A 4% dioxane solution of lactic acid-caprolactone copolymer (P (LA / CL); polymerization ratio 50:50) is prepared.
-Put the PLLA cloth on a 20cm x 20cm glass mold and pour the P (LA / CL) solution there.
-Place the sponge obtained in 3 above on it, quickly transfer it to a -80 ° C freezer and freeze it.
・ Heat from -40 ° C to 40 ° C at a constant rate over 12 hours under vacuum reduced pressure (0.01mmHg), further dry at 40 ° C under vacuum reduced pressure for 6 hours, and further at 70 ° C under vacuum reduced pressure. Dry for 12 hours to obtain a complex of collagen sponge and PLLA cloth and P (LA / CL) sponge.
[0011]
[Example 2]
1. Preparation of lactic acid-caprolactone reinforcing material (cloth)-Fabricate a lactic acid-caprolactone copolymer (P (LA / CL); polymerization ratio 75:25) fiber. (Organization: 7 types: milling fabric, double-sided knitting fabric, twin knitted fabric, plain woven fabric, tengu knitted fabric, non-woven fabric)
・ Plasma treatment (oxygen, 100 W, 0.5 torr, 30 seconds) of P (LA / CL) cloth to make it hydrophilic. 2. Complexed with collagen sponge ・ Atelocollagen (Nitta gelatin) is used as a raw material to prepare a 3 mg / ml, pH 3.0 collagen aqueous solution.
Add 0.5 g of chloroform to 50 g of this solution, and vigorously stir for 1 minute at 6000 rpm using a homogenizer.
-Place the hydrophilic P (LA / CL) cloth on an aluminum mold (11cm x 8.5cm) and pour the resulting creamy collagen foam.
-Let stand for 1 hour in a -40 ° C freezer and freeze.
-Heated at a constant rate from -40 ° C to 40 ° C for 12 hours under vacuum reduced pressure (0.01mmHg), and further dried at 40 ° C under vacuum reduced pressure for 12 hours to reinforce P (LA / CL) A collagen sponge complexed with is obtained.
3. Thermal cross-linking The above composite is heat-dried at 105 ° C. for 24 hours under vacuum under reduced pressure to be thermally cross-linked.
4). Chemical cross-linking The above composite is immersed in a 0.2 wt% glutaraldehyde / acetic acid solution at 5 ° C. for 24 hours for chemical cross-linking.
-Wash the resulting sponge thoroughly with ion-exchanged water.
-After washing, immerse in 15% ethanol aqueous solution.
・ Leave in a freezer at -135 ° C for 12 hours to freeze.
-Heating at a constant rate from -40 ° C to 40 ° C under vacuum reduced pressure (0.01 mmHg) over 12 hours, and further drying at 40 ° C under vacuum reduced pressure for 12 hours to obtain a chemically crosslinked complexed collagen sponge.
[0012]
(Transplant to animals)
Rabbit bone marrow was collected, mixed with DMEM medium containing 15% fetal bovine serum, and cultured for about 1 week. Further, the cells were subcultured for about 1 week and grown to about 10 6 . The cells were seeded on a composite of the milled knitted fabric obtained in Example 2 and a cross-linked collagen sponge obtained by cutting into a cylindrical shape having a diameter of 4 mm and a thickness of 3 mm. The rabbit's knee joint was exposed, a 4 mm diameter and 4 mm deep osteochondral defect was created using a drill, and a cell seeded sample was inserted into the defect. Rabbits were sacrificed 1, 2, and 4 weeks after the operation, and tissue sections of the transplanted sites were prepared. After the operation, regeneration of the cartilage tissue at the joint surface and regeneration of the bone tissue at the bone defect were observed with time.
[0013]
【The invention's effect】
In the present invention, the cross-linking of collagen and the reinforcing effect by the reinforcing material can improve handling convenience, can be cut into a free shape and size, can be added with sufficient suturing strength, and the reinforcing material can be absorbed. Since the reinforcing material is a porous body (with moderately open eyes), it has many features and effects such as nutrition permeation.
Therefore, it can be used not only for cartilage regeneration, but also as a substrate for regeneration of other organs, for example, as a substrate for cultured skin. In particular, a three-layer type having a sandwich structure can be suitably used as a cultured skin base material.
Claims (13)
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WO2006021992A1 (en) * | 2004-08-24 | 2006-03-02 | Gunze Limited | Process for producing collagen sponge, process for producing artificial skin, artificial skin and cell tissue culture substrate |
JP2011078710A (en) * | 2009-09-08 | 2011-04-21 | Rie Tsuchiya | Implantation material for cartilage |
KR101628821B1 (en) * | 2015-03-02 | 2016-06-13 | 강원대학교산학협력단 | Biocompatible Solubilized Scaffold Extract Derived from Decellularized Organ Tissue, Method for Preparating the Biocompatible Scaffold Extract and Uses for Thereof |
WO2018123814A1 (en) * | 2016-12-28 | 2018-07-05 | 株式会社高研 | High-strength collagen sponge |
JP2021132881A (en) * | 2020-02-27 | 2021-09-13 | グンゼ株式会社 | Cartilage regeneration substrate |
EP4119169A1 (en) * | 2021-07-16 | 2023-01-18 | Lietuvos Sveikatos Mokslu Universitetas | A construct for articular cartilage regeneration and a method of preparation |
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