WO2007066650A1 - Culture substrate and culture method for cartilage tissue regeneration - Google Patents

Culture substrate and culture method for cartilage tissue regeneration Download PDF

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WO2007066650A1
WO2007066650A1 PCT/JP2006/324241 JP2006324241W WO2007066650A1 WO 2007066650 A1 WO2007066650 A1 WO 2007066650A1 JP 2006324241 W JP2006324241 W JP 2006324241W WO 2007066650 A1 WO2007066650 A1 WO 2007066650A1
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culture
substrate
cartilage tissue
chitosan
culture substrate
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Tokifumi Majima
Norimasa Iwasaki
Kazuo Harada
Sachiko Nonaka
Yasuhiko Kasahara
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Chemical Biology Institute
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0655—Chondrocytes; Cartilage
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    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • 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
    • A61L27/56—Porous materials, e.g. foams or sponges
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/72—Chitin, chitosan

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  • the thus-produced cartilage tissue-attached to the light-colored, acidic biopolymer-containing material, which is produced in this manner, can be suitably used for wound healing.

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Abstract

It is intended to provide a substrate for culturing a cartilage cell and a culture method to be used for cartilage tissue regeneration. The object can be achieved by a culture substrate which is a culture substrate for cartilage tissue regeneration containing a fabric or a knit comprising a chitosan/acidic biological polymer hybrid fiber in which the inside of the fiber comprises chitosan or a salt thereof and the surface of the fiber is covered with a composite of chitosan and an acidic biological polymer selected from the group consisting of hyaluronic acid, chondroitin sulfate and dermatan sulfate, and in which the space between threads of the fabric or the knit is 250 to 500 μm and the porosity of the inside of the substrate is 65 to 94%.

Description

明 細 書 Specification
軟骨組織再生用培養基材および培養方法 Culture substrate and culture method for cartilage tissue regeneration
技術分野 Technical field
[0001] 本発明は、変形性関節症などの疾患や事故などの原因により損傷した軟骨組織を 再生させるために用いられる移植用軟骨組織の培養のための培養基材、および該 基材を用いる軟骨細胞培養方法に関するものである。 [0001] The present invention provides a culture substrate for culturing cartilage tissue for transplantation used to regenerate cartilage tissue damaged due to diseases such as osteoarthritis or accidents, and the use of the substrate. The present invention relates to a method for culturing chondrocytes.
背景技術 Background technology
[0002] 高齢化社会を迎え、関節疾患患者は人口の 1 %にも達しようとして!/、る。これらの疾 患者の大部分は軟骨組織が損傷を受け、壊死することによって起こる変形性関節症 や慢性リウマチによるものと言われている。軟骨は自己再生能がきわめて低いことか ら、外科的治療が必要な場合には人工関節置換術が施されている。しかし、この方 法では装着物力 の金属イオンの溶出による炎症や骨接合部との緩み、耐久性等に 大きな課題があり、医療用具としての寿命は 10年程度とされていることから、人工関 節置換術は根治的な治療法にはなって!/、な 、。 [0002] As we enter an aging society, patients with joint diseases are on the verge of reaching 1% of the population! Most patients with these diseases are said to be suffering from osteoarthritis or chronic rheumatism, which is caused by damage to cartilage tissue and necrosis. Cartilage has extremely low self-renewal ability, so when surgical treatment is necessary, artificial joint replacement is performed. However, this method has major issues such as inflammation due to the elution of metal ions from the implant, loosening of the joint with the bone, and durability, and the lifespan of the device as a medical device is estimated to be about 10 years. Replacement surgery is not a definitive treatment!
[0003] そこで、近年、自己再生能の低 、組織を対象に、正常部位の自己組織を一度体外 に取り出し、培養して増殖'分化させた後、再び体内に移植して目的組織の再生を 図ろうとする再生医療の研究が盛んに行われて 、る。細胞はある物質の表面に接着 することによって増殖'分ィ匕が起こることから、再生医療の発展には培養細胞が接着 するための足場となる良好な人工基材の開発が、不可欠とされている。 [0003] Therefore, in recent years, attempts have been made to target tissues with low self-regeneration ability, by removing autologous tissues from normal parts of the body, culturing them, proliferating and differentiating them, and then transplanting them back into the body to regenerate the target tissue. There is a lot of research going on into regenerative medicine. Cells proliferate when they adhere to the surface of a certain material, so the development of good artificial substrates that serve as scaffolds for cultured cells to adhere to is essential for the development of regenerative medicine. There is.
[0004] 一般に、組織再生用の基材に求められる条件として、（1)炎症反応が見られず、生 体親和性に優れていること、（2)生体吸収性であること、（3)細胞の接着性がよいこと 、（4)細胞の活性を維持できること、（5)細胞の増殖 ·分ィ匕による組織再生が可能な 3 次元構造を有することが挙げられる。さらに、股関節部位の軟骨には 20Mpa程度ま での圧縮応力がかかることから、上記 5つの条件以外に（6)体内で組織が再生される までの形状の安定性が確保されること、および (7)機械的強度を有することの 2つの 条件も同時に満たす基材の開発が必要と考えられる。 [0004] In general, the conditions required for a base material for tissue regeneration are (1) no inflammatory reaction and excellent biocompatibility, (2) bioabsorbability, and (3) (4) ability to maintain cell activity; and (5) three-dimensional structure that allows tissue regeneration through cell proliferation and division. Furthermore, since compressive stress of up to approximately 20 MPa is applied to the cartilage in the hip joint, in addition to the above five conditions, (6) shape stability must be ensured until the tissue is regenerated within the body; 7) It is considered necessary to develop a base material that simultaneously satisfies the two conditions of having mechanical strength.
[0005] これまでに軟骨細胞培養基材として、ポリグリコール酸やポリ乳酸等の生体吸収性 の合成高分子を用 、たファイバーやスポンジが検討されてきた (特公平 6— 6155号 、特表平 8— 511679号、 Ito, K.等， Mat. Res. Soc. Proc, 252, 359—365 (1992), Fr eed, L. E.等， J. Biol. Mater. Res., 27, 11-23 (1993)等）。これらの合成高分子材料 は体内での加水分解物が生体内の代謝中間体と同一であるため毒性がなぐ高重 合体が得られるため機械的強度を有し、成形が容易であるなどの長所がある。しかし 、これらの合成高分子は細胞の接着性に欠けるため、材料表面に生体の細胞接着 性因子である RGD (アルギニン一グリシンーァスパラギン酸のトリペプチド）や、ゼラ チン、コラーゲン等の生体高分子を固定ィ匕する方法によって細胞の接着性を向上さ せることが検討されてきた（Yamaoka, T.等， J. Biol. MacromoL, 25, 265-271 (1999) 等)。しかし、このような化学的固定方法は操作が煩雑であり、また、固定化処理に使 用した薬品の残存等も懸念される。 [0005] So far, bioabsorbable materials such as polyglycolic acid and polylactic acid have been used as chondrocyte culture substrates. Fibers and sponges using synthetic polymers have been investigated (Japanese Patent Publication No. 6-6155, Japanese Patent Publication No. 8-511679, Ito, K. et al., Mat. Res. Soc. Proc, 252, 359). 365 (1992), F eed, LE et al., J. Biol. Mater. Res., 27, 11-23 (1993), etc.). These synthetic polymer materials have advantages such as the fact that the hydrolyzate in the body is the same as the metabolic intermediate in the body, so a non-toxic high polymer can be obtained, which has mechanical strength and is easy to mold. There is. However, since these synthetic polymers lack cell adhesion, they are coated with biological cell adhesion factors such as RGD (arginine-glycine-aspartic acid tripeptide), gelatin, and collagen on the material surface. Improving cell adhesion by immobilizing polymers has been investigated (Yamaoka, T. et al., J. Biol. MacromoL, 25, 265-271 (1999), etc.). However, such chemical fixation methods are complicated to operate, and there are also concerns about residual chemicals used in the fixation process.
[0006] 一方、天然高分子を用いた軟骨細胞培養用基材としては、コラーゲンのゲルゃス ポンジ状基材（特開平 6-22744号公報、特表平 9-510639号公報、特開 2001-224678 号公報、 Fujisato, T.等， Biomaterials, 17,155-162 (1996)等）、キチンやキトサンのス ポンジ状基材が検討されてきた（Park, Y. J.等， Biomaterials, 21, 153-159 (2000)等 )。これらの基材は細胞接着性には優れるが、形状安定性および機械的強度に問題 がある。また、コラーゲンは原材料費が高価であるとともに、抗原性や BSE等の感染も 懸念される。 [0006] On the other hand, as substrates for chondrocyte culture using natural polymers, collagen gel and sponge-like substrates (Japanese Patent Application Laid-open No. 6-22744, Japanese Patent Publication No. 9-510639, Japanese Patent Application Laid-Open No. 2001-2001) -224678, Fujisato, T. et al., Biomaterials, 17, 155-162 (1996), etc.), chitin and chitosan sponge-like substrates have been investigated (Park, Y. J. et al., Biomaterials, 21, 153-159 ( 2000) etc.). Although these substrates have excellent cell adhesion, they have problems with shape stability and mechanical strength. In addition, collagen is expensive as a raw material, and there are concerns about antigenicity and infections such as BSE.
[0007] 酸性高分子としてアルギン酸を主原料に塩基性高分子としてキトサンを用いた基材 [0007] Base material using alginic acid as the main raw material as an acidic polymer and chitosan as a basic polymer
(特許第 3616344号)やキトサンとヒアルロン酸等の酸性生体高分子力もなるハイブ リツド繊維を用いた培養基材 (WO 2004Z003130)に関する報告がある。しかし、 この培養基材は繊維の製造方法等の詳細な記載はあるが、基材の具体的な構造に つ!、ては明記されて!、な!/、。 (Patent No. 3616344) and a culture substrate using hybrid fibers that also have acidic biopolymers such as chitosan and hyaluronic acid (WO 2004Z003130). However, although there is a detailed description of the fiber manufacturing method for this culture substrate, the specific structure of the substrate is not specified!, na!/,.
[0008] これまでに基材の構造に関しては、軟骨細胞の増殖および組織再生の足場として 用いられる天然高分子多孔質構造体の細孔は 20〜： L00 μ mが好ま 、との報告（ 特開 2003— 10309)や、細孔の大きさが 100〜200 /ζ πιの難水溶性ヒアルロン酸ゲ ルが細胞を保持するのに好ましいとの報告がある（特開 2003— 10308)。し力し、細 胞を保持することのみを考えた場合にはこの程度の小孔が好ましいと考えられるが、 軟骨細胞が増殖 ·分ィ匕し、組織を形成する際にはこのような小孔では細胞外マトリツ タスを産生するための空間が確保できず、良好な軟骨組織の形成には不適当なこと が予想される。 [0008] Regarding the structure of the base material, it has been reported that the pore size of natural polymer porous structures used as scaffolds for the proliferation of chondrocytes and tissue regeneration is preferably 20 to 100 μm (specifically There is a report that a poorly water-soluble hyaluronic acid gel with a pore size of 100 to 200 /ζ πι is preferable for retaining cells (Japanese Patent Application Laid-open No. 2003-10308). This size of pores is considered preferable if we are only concerned with holding the cells. When chondrocytes proliferate and divide to form tissue, such small pores cannot secure space for producing extracellular matrices, which is inappropriate for the formation of good cartilage tissue. is expected.
[0009] 一方、軟骨細胞の培養方法に関してもこれまでにいくつかの検討が行われている。 [0009] On the other hand, several studies have been conducted regarding methods for culturing chondrocytes.
軟骨組織は歩行などの日常的な動作により 5〜： LOMPa程度の静水圧を受けている とされている。また、軟骨細胞は強力な血管新生阻害作用をもち、軟骨組織への血 管の進入を抑制していることから、軟骨組織は酸素分圧が低いと考えられている。そ こで、これらの生体条件を模擬した培養方式として静水圧を負荷する方式 (Parkkinen , J.等， Arch. Biochem. Biophys., 300, 458—465 (1993)、 Mizuno, S.等， Material Scie nce & Engineering C, c6, 301-306 (1998)等）や、低酸素下で培養する方式（Domm, C.等， Osteoarthritis Cartilage, 10, 13-22 (2002)等）などが研究され、報告されてい る。また、培地を撹拌する方式についても報告（Freed, L. E.等， J. Cell Biochem., 51 , 257-264 (1993))がある力 これらの培養方式と基材形状との関係については明ら かとなつていない。 Cartilage tissue is said to be subjected to hydrostatic pressure of approximately 5 to LOMPa due to daily activities such as walking. Additionally, chondrocytes have a strong angiogenesis inhibitory effect and inhibit the entry of blood vessels into cartilage tissue, so it is thought that cartilage tissue has a low oxygen partial pressure. Therefore, a method of applying hydrostatic pressure as a culture method that simulates these biological conditions (Parkkinen, J. et al., Arch. Biochem. Biophys., 300, 458-465 (1993), Mizuno, S. et al., Material Science & Engineering C, c6, 301-306 (1998), etc.) and methods of culturing under hypoxia (Domm, C. et al., Osteoarthritis Cartilage, 10, 13-22 (2002), etc.) have been studied. It has been reported. There is also a report on the method of stirring the culture medium (Freed, L. E. et al., J. Cell Biochem., 51, 257-264 (1993)).The relationship between these culture methods and the shape of the substrate is not clear. I'm not used to it.
発明の開示 Disclosure of invention
発明が解決しょうとする課題 Problems that the invention seeks to solve
[0010] 本発明は、培養基材を用いた生体外での培養によって軟骨組織の主要構成成分 であるタイプ IIコラーゲンが基材内部まで形成され、且つ、基材の占める割合が非常 に低 、軟骨組織を得ることが可能であって、軟骨組織の形成とともに機械的強度も 増加し、生体内への移植後も良好な軟骨組織の再生が起こる培養基材を提供するこ とを目的とする。 [0010] The present invention is characterized in that type II collagen, which is a main component of cartilage tissue, is formed inside the substrate by culturing in vitro using a culture substrate, and that the proportion occupied by the substrate is extremely low. The purpose of the present invention is to provide a culture substrate from which cartilage tissue can be obtained, mechanical strength increases with the formation of cartilage tissue, and good regeneration of cartilage tissue occurs even after transplantation into a living body. .
課題を解決するための手段 Means to solve problems
[0011] 本発明は、繊維内部がキトサンまたはその塩よりなり、繊維表面がキトサンとヒアル ロン酸、コンドロイチン硫酸、およびデルマタン硫酸よりなる群カゝら選択される酸性生 体高分子との複合体で被覆されているキトサン Ζ酸性生体高分子ハイブリッド繊維よ りなる織物または編物を含む軟骨組織再生用培養基材であって、構造物内部の空 隙率が 65〜94%であり、織物または編物の糸と糸の間隙は 250〜500 μ mである 培養基材を要旨とする。 [0012] 培養基材に占める空隙率が低い場合、即ち、培養基材に占める基材の割合が高 い場合には、培養中に軟骨組織が形成されるための空間が確保できない。また、移 植後生体内で基材が分解されるのに時間がかかり、基材部分における組織形成能 が低くなる恐れがある。軟骨組織には血管が発達して 、な 、ために代謝も遅 、ことか ら、良好な軟骨組織の再生には基材の分解速度も考慮した適正な 3次元の構造が 非常に重要と考えられる。さらに、培養基材に占める空隙率が高すぎる場合、即ち、 培養組織に占める基材の割合が低 ヽ場合には、培養時に播種した軟骨細胞が付着 し増殖、分ィ匕するための足場となる基材が少なすぎるため、細胞密度が低下してしま い、組織の形成が起こり難くなる。 [0011] The present invention is characterized in that the interior of the fiber is made of chitosan or a salt thereof, and the surface of the fiber is a composite of chitosan and an acidic biopolymer selected from the group consisting of hyaluronic acid, chondroitin sulfate, and dermatan sulfate. A culture substrate for cartilage tissue regeneration comprising a woven or knitted fabric made of coated chitosan Ζ acidic biopolymer hybrid fiber, in which the porosity inside the structure is 65 to 94%, and the porosity of the woven or knitted fabric is 65 to 94%. The gap between the threads is 250 to 500 μm.The culture substrate is essential. [0012] When the porosity of the culture substrate is low, that is, when the proportion of the substrate in the culture substrate is high, it is not possible to secure a space for cartilage tissue to form during culture. Furthermore, it takes time for the base material to decompose in vivo after transplantation, and there is a risk that the tissue forming ability in the base material portion may be reduced. Cartilage tissue has developed blood vessels and therefore has a slow metabolism. Therefore, we believe that an appropriate three-dimensional structure that also takes into account the decomposition rate of the base material is extremely important for good cartilage tissue regeneration. It will be done. Furthermore, if the porosity of the culture substrate is too high, that is, if the proportion of the substrate in the cultured tissue is low, the chondrocytes seeded during culture may attach, proliferate, and divide into scaffolds. Because there is too little substrate to form, cell density decreases, making it difficult for tissue formation to occur.
[0013] 織物または編物の糸と糸の間隙が小さすぎると、基材内部までの十分な培地移動 が起こりづらいために、基材内部まで軟骨組織を形成させることが困難となる。また、 織物または編物の糸と糸の間隙が大きすぎると、培養中に基材の変形などが起こり、 形態の維持が困難となる。 [0013] If the gap between the threads of the woven or knitted material is too small, sufficient movement of the medium to the inside of the base material is difficult to occur, making it difficult to form cartilage tissue inside the base material. Furthermore, if the gaps between the threads of the woven or knitted material are too large, the substrate may deform during culture, making it difficult to maintain the shape.
[0014] 本発明はまた、上記培養基材を用いた生体外での軟骨細胞の培養方法にも関す る。 [0014] The present invention also relates to a method for culturing chondrocytes in vitro using the above culture substrate.
本発明はさらに上記培養方法で得られた移植用軟骨組織にも関する。 発明の効果 The present invention further relates to a cartilage tissue for transplantation obtained by the above culture method. Effect of the invention
[0015] 本発明の培養基材を用いた生体外での培養によって軟骨組織の主要構成成分で あるタイプ IIコラーゲンが基材内部まで形成され、且つ、基材の占める割合が非常に 低い軟骨組織を得ることが可能であって、軟骨組織の形成とともに機械的強度も増 加し、生体内への移植後も良好な軟骨組織の再生が起こる培養基材が得られる。 図面の簡単な説明 [0015] By culturing in vitro using the culture substrate of the present invention, type II collagen, which is the main component of cartilage tissue, is formed inside the substrate, and the proportion of the substrate is extremely low. It is possible to obtain a culture substrate whose mechanical strength increases with the formation of cartilage tissue, and which allows good regeneration of cartilage tissue even after transplantation into a living body. Brief description of the drawing
[0016] [図 1]本発明の培養基材の構造の例を示す模式図である。 [0016] FIG. 1 is a schematic diagram showing an example of the structure of the culture substrate of the present invention.
[図 2]異なる間隙の織物力もなる培養基材を用いて培養した場合の生成したタンパク 質量の比較を示すグラフである。各値は平均値と標準誤差で示した (n= 5)。 [Figure 2] A graph showing a comparison of the amount of protein produced when cultured using culture substrates with different interstitial weaving forces. Each value is expressed as the mean and standard error (n= 5).
[図 3]異なる間隙の織物力もなる培養基材を用いて培養した場合の生成した酸性ムコ 多糖量の比較を示すグラフである。各値は平均値と標準誤差で示した (n= 5)。 [Figure 3] A graph showing a comparison of the amount of acidic mucopolysaccharide produced when culturing using culture substrates with different interstitial weaving forces. Each value is expressed as the mean and standard error (n= 5).
[図 4]異なる構造の織物力もなる培養基材を用いて培養した場合の生成したタンパク 質量の経時変化を示すグラフである。各値は平均値と標準誤差で示した (n= 5)。 圆 5]異なる構造の織物力もなる培養基材を用いて培養した場合の生成した酸性ムコ 多糖量の経時変化を示すグラフである。各値は平均値と標準誤差で示した (n= 5)。 圆 6]回転培養を行った培養組織の湿重量の経時変化を示すグラフである。培養開 始 1週間後から回転培養した。重量には基材を含む。各値は平均値と標準誤差で示 した (n= 5)。 [Figure 4] Proteins produced when cultured using culture substrates with different structures that also have weaving strength. It is a graph showing a change in mass over time. Each value is expressed as the mean and standard error (n= 5).圆5] This is a graph showing changes over time in the amount of acidic mucopolysaccharide produced when culturing using culture substrates with different structures that also have weaving strength. Each value is expressed as the mean and standard error (n= 5).圆6] This is a graph showing changes over time in the wet weight of cultured tissues subjected to rotational culture. One week after the start of culture, rotation culture was carried out. Weight includes base material. Each value is expressed as the mean and standard error (n= 5).
圆 7]回転培養を行った培養組織のタンパク質量の経時変化を示すグラフである。培 養開始 1週間後から回転培養した。各値は平均値と標準誤差で示した (n= 3)。 圆7] This is a graph showing changes over time in the amount of protein in cultured tissues subjected to rotational culture. Rotation culture was started one week after the start of culture. Each value is expressed as the mean and standard error (n= 3).
[図 8]回転培養を行った培養組織の DNA量の経時変化を示すグラフである。培養開 始 1週間後から回転培養した。各値は平均値と標準誤差で示した (n= 3)。 [Figure 8] A graph showing changes over time in the amount of DNA in cultured tissues subjected to rotational culture. One week after the start of culture, rotation culture was carried out. Each value is expressed as the mean and standard error (n= 3).
圆 9]回転培養を行った際のタイプ IIコラーゲンの染色像を示す写真のコピーである 圆 10]回転培養と低酸素条件下での培養を組み合わせた際の湿重量の変化を示す グラフである。培養開始 1週間後から回転培養、 2週間後から低酸素下で培養した。 各値は平均値と標準誤差で示した (n= 3)。 圆 9] This is a copy of a photograph showing the staining image of type II collagen during rotational culture. 圆 10] A graph showing the change in wet weight when combining rotational culture and culture under hypoxic conditions. . One week after the start of culture, the cells were cultured by rotation, and two weeks later, they were cultured under hypoxia. Each value is expressed as the mean and standard error (n= 3).
圆 11]回転培養と低酸素条件下での培養を組み合わせた際のタンパク質量の変化 を示すグラフである。培養開始 1週間後から回転培養、 2週間後から低酸素下で培養 した。各値は平均値と標準誤差で示した (n= 3)。 圆11] This is a graph showing the change in protein amount when rotary culture and culture under hypoxic conditions are combined. One week after the start of culture, the cells were cultured in rotation, and two weeks later, they were cultured under hypoxia. Each value is expressed as the mean and standard error (n= 3).
圆 12]回転培養と低酸素条件下での培養を組み合わせた際の DNAの変化を示す グラフである。培養開始 1週間後から回転培養、 2週間後から低酸素下で培養した。 各値は平均値と標準誤差で示した (n= 3)。 圆12] This is a graph showing changes in DNA when rotational culture and culture under hypoxic conditions are combined. One week after the start of culture, the cells were cultured by rotation, and two weeks later, they were cultured under hypoxia. Each value is expressed as the mean and standard error (n= 3).
圆 13]回転培養と低酸素条件下での培養を組み合わせて行った際のタイプ IIコラー ゲン染色像を示す写真のコピーである。 圆13] This is a copy of a photograph showing type II collagen staining when a combination of rotary culture and culture under hypoxic conditions was performed.
圆 14]回転培養と低酸素条件下での培養を組み合わせて行った際の培養組織の硬 度変化を示すグラフである。 圆14] This is a graph showing changes in the hardness of cultured tissue when a combination of rotational culture and culture under hypoxic conditions was performed.
圆 15]培養基材 [2]および [3]を用いて回転培養を行った際の湿重量の変化を示す グラフである。 1週間の静置培養後、回転培養を行った。 圆15] This is a graph showing the change in wet weight when rotary culture was performed using culture substrates [2] and [3]. After one week of static culture, rotational culture was performed.
圆 16]培養基材 [2]および [3]を用いて回転培養を行った際のタンパク質量の変化 を示すグラフである。 1週間の静置培養後、回転培養を行った。 圆16] Changes in protein amount during rotational culture using culture substrates [2] and [3] This is a graph showing. After one week of static culture, rotational culture was performed.
[図 17]培養基材 [2]および [3]を用いて回転培養を行った際の DNA量の変化を示 すグラフである。 1週間の静置培養後、回転培養を行った。 [Figure 17] A graph showing changes in the amount of DNA during rotational culture using culture substrates [2] and [3]. After one week of static culture, rotational culture was performed.
[図 18]培養基材 [3]を用いて回転培養を行った際のタイプ IIコラーゲン染色像を示す 写真のコピーである。 [Figure 18] This is a copy of a photograph showing a type II collagen staining image when rotational culture was performed using the culture substrate [3].
[図 19]培養基材 [2]を用いて骨髄幹細胞力も軟骨細胞への分ィ匕誘導を行った際の タイプ IIコラーゲン染色像を示す写真のコピーである。 [Figure 19] This is a copy of a photograph showing a type II collagen staining image when bone marrow stem cells were induced to differentiate into chondrocytes using the culture substrate [2].
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
[0017] 繊維内部がキトサンまたはその塩よりなり、繊維表面がキトサンとヒアルロン酸、コン ドロイチン硫酸、およびよりなる群から選択される酸性生体高分子との複合体で被覆 されているキトサン Z酸性生体高分子ハイブリッド繊維は、 WO 2004/003130^- 公報に記載の方法により製造できる。 [0017] Chitosan Z acidic biomolecule, in which the interior of the fiber is made of chitosan or a salt thereof, and the fiber surface is coated with a complex of chitosan, hyaluronic acid, chondroitin sulfate, and an acidic biopolymer selected from the group consisting of The polymer hybrid fiber can be produced by the method described in WO 2004/003130^-.
即ち、 1つの製造方法では、 1)キトサンを酸の水溶液に溶解しキトサンの塩の水溶 液を調製し； 2)キトサンの塩の水溶液を、アルカリ土類金属の塩を凝固剤として用い て湿式紡糸して繊維を形成させ; 3)その繊維を生体吸収性の酸性生体高分子の溶 液に浸漬して、繊維表面でキトサンと酸性生体高分子を反応させてキトサン Z酸性 生体高分子ハイブリッド繊維を形成させ; 4)場合によりハイブリッド繊維を延伸し; 5) ノ、イブリツド繊維を塩基、 2塩基酸以上の無機酸もしくはその塩、または 3塩基酸以上 の有機酸もしくはその塩の水溶液で処理する。 That is, in one production method, 1) chitosan is dissolved in an aqueous acid solution to prepare an aqueous solution of a chitosan salt; 2) an aqueous solution of a chitosan salt is wet-processed using an alkaline earth metal salt as a coagulant. 3) The fibers are immersed in a solution of a bioabsorbable acidic biopolymer to react with chitosan and the acidic biopolymer on the fiber surface to form a chitosan Z acidic biopolymer hybrid fiber. 4) optionally drawing the hybrid fiber; 5) treating the hybrid fiber with an aqueous solution of a base, an inorganic acid with a dibasic acid or more or a salt thereof, or an organic acid with a tribasic acid or more or a salt thereof; .
[0018] 他の製造方法では、 1)キトサンを酸の水溶液に溶解しキトサンの塩の水溶液を調 製し； 2)キトサンの塩の水溶液を、塩基、 2塩基酸以上の無機酸もしくはその塩、また は 3塩基酸以上の有機酸もしくはその塩を凝固剤として用いて湿式紡糸して繊維を 形成させ; 3)その繊維を生体吸収性の酸性生体高分子の溶液に浸漬して、繊維表 面でキトサンと酸性生体高分子を反応させてキトサン Z酸性生体高分子ハイブリッド 繊維を形成させ; 4)場合によりハイブリッド繊維を延伸する。 [0018] In another production method, 1) chitosan is dissolved in an aqueous acid solution to prepare an aqueous solution of a chitosan salt; 2) the aqueous solution of a chitosan salt is dissolved in a base, an inorganic acid of dibasic acid or more, or a salt thereof; , or wet spinning using an organic acid with three or more basic acids or its salt as a coagulant to form fibers; 3) immersing the fibers in a solution of bioabsorbable acidic biopolymers to coat the fiber surface. Chitosan and acidic biopolymer are reacted on the surface to form chitosan Z acidic biopolymer hybrid fiber; 4) Optionally, the hybrid fiber is stretched.
[0019] このハイブリッド繊維は、（1)炎症反応が見られず、生体親和性に優れていること、 ( 2)生体吸収性であること、（3)細胞の接着性がよいこと、（4)細胞の活性を維持でき ること等の特徴を有する。 [0020] このようにして製造した繊維を用いて、公知の方法により、織物、編物、糸、綿状物 を製造する。ここで「糸」とは上のように製造した繊維を撚糸して得られるものを言い、 「綿状物」とは上のように製造した直径 10〜50 mの繊維を塊状にしたものを言う。 織物または編物の場合、糸と糸の間の間隙を 250〜500 mとすることが好ましい。 [0019] This hybrid fiber has the following properties: (1) No inflammatory reaction and excellent biocompatibility, (2) Bioabsorbability, (3) Good cell adhesion, (4) ) It has characteristics such as being able to maintain cell activity. [0020] Using the thus produced fibers, woven fabrics, knitted fabrics, threads, and cotton-like materials are produced by known methods. Here, "thread" refers to the material obtained by twisting the fibers produced as above, and "cotton" refers to the fibers produced as above that are made into a lump with a diameter of 10 to 50 m. To tell. In the case of woven or knitted fabrics, the gap between the threads is preferably 250 to 500 m.
[0021] 本発明の培養基材のー態様によれば、上記織物または編物を、 2枚以上重ね合わ せ、上記の糸を用いて固定して形態を保つ。その場合、培養基材における基材全体 の空隙率が 60%を超えるようにすることが好ま U、。 [0021] According to an embodiment of the culture substrate of the present invention, two or more of the above-mentioned woven or knitted fabrics are overlapped and fixed using the above-mentioned thread to maintain the shape. In that case, it is preferable that the porosity of the entire culture substrate exceeds 60%.
[0022] 本発明の培養基材の他の態様によれば、織物または編物の間に、上記綿状物が 挟まれており、各織物または編物間を上記の糸で固定されて形態を保つ。 [0022] According to another aspect of the culture substrate of the present invention, the cotton-like material is sandwiched between woven or knitted fabrics, and the woven or knitted fabrics are fixed with the above-mentioned threads to maintain their shape. .
[0023] 本発明の培養基材のさらなる態様によれば、帯状の織物または編物を丸めて円筒 形にし、上記の糸で固定し、またはキトサン溶液を塗布し、乾燥させて固定して形態 を保ち、円筒内部に上記綿状物が充填される。 [0023] According to a further aspect of the culture substrate of the present invention, a strip-shaped woven or knitted fabric is rolled up into a cylindrical shape and fixed with the above-mentioned threads, or a chitosan solution is applied, dried and fixed, and the shape is changed. The inside of the cylinder is filled with the flocculent material.
[0024] V、ずれの場合も培養基材の空隙率が 60〜96%、好ましくは 65〜94%、特に好ま しくは 80〜94%となることが必要である。培養基材の「空隙率」とは（1—繊維の体積 Z基材の体積) xlOOをいう。空隙率の測定は次のようにして行う。基材の重量を測定 し、繊維の密度 1. 013gZcm3から基材中の繊維の体積を算出する。また基材の体 積を測定し、これらの値をもとに、上式より空隙率を算出する。 [0024] In the case of V and displacement, it is necessary that the porosity of the culture substrate is 60 to 96%, preferably 65 to 94%, and particularly preferably 80 to 94%. The “porosity” of the culture substrate is (1—volume of fibers Z volume of substrate) xlOO. The porosity is measured as follows. Measure the weight of the base material and calculate the volume of the fibers in the base material from the fiber density 1.013gZcm3 . The volume of the base material is also measured, and based on these values, the porosity is calculated using the above formula.
[0025] 上記の培養基材を用いる動物細胞の培養は通常の動物細胞培養法 (例えば、 Kla gsourn, M., Large ¾caie Preparation of Chondrocytes , Methods in iinzymoi., 58:5 60(1979)を参照）に準じて行う。先ず、予め、該培養基材をオートクレープで加熱滅 菌するか、ガス殺菌等を行い形状 '特性が壊れないように殺菌処理を施し、殺菌した 培地に添加する。次に、動物細胞を培養基材上にできるだけ 3次元的に均一に播ぃ て培養する。培養に使用する細胞としてはゥサギ、ゥシ、ゥマ、ィヌ、ネコ、ヒト等の哺 乳動物由来の細胞であり、軟骨細胞または軟骨細胞になりうる幹細胞であれば、い ずれの細胞でも培養可能である。好ましい細胞は、ヒト由来のものであり、特に好まし V、のは移植しょうとする患者由来の軟骨細胞または幹細胞である。 [0025] Cultivation of animal cells using the above-mentioned culture substrate can be carried out using conventional animal cell culture methods (see, for example, Kla gsourn, M., Large ¾caie Preparation of Chondrocytes, Methods in iinzymoi., 58:5 60 (1979)). ). First, the culture substrate is sterilized in advance by heat sterilization in an autoclave or gas sterilization so as not to destroy its shape and characteristics, and then added to the sterilized medium. Next, animal cells are spread on the culture substrate as uniformly as possible in three dimensions and cultured. The cells used for culture are cells derived from mammals such as rabbits, cows, horses, dogs, cats, and humans, and any chondrocytes or stem cells that can become chondrocytes can be used. Can be cultured. Preferred cells are of human origin, and particularly preferred are chondrocytes or stem cells derived from the patient to whom the transplant is to be performed.
[0026] 培地としては、通常の動物細胞培養法で用いられるもの、例えばヒト血清を含む D MEM (Dulbecco' s Modified Eagle' s Medium)などが使用出来る。培地にはいずれ かの成長因子、例えば TGF |8 (トランスフォーミング成長因子 j8 )、 FGF (線維芽細 胞増殖因子）， ChM- 1 (コンドロモジエリン一 1)などを添カ卩してもよ!、。 [0026] As the medium, those used in normal animal cell culture methods, such as DMEM (Dulbecco's Modified Eagle's Medium) containing human serum, can be used. The medium will eventually You can also add growth factors such as TGF|8 (transforming growth factor j8), FGF (fibroblast growth factor), ChM-1 (chondromodielin-1), etc.
[0027] 播種した細胞が培養基材上で良好に増殖、分化するためには細胞付着 ·吸着性の 高 、培養基材は極めて重要である。 [0027] In order for the seeded cells to proliferate and differentiate favorably on the culture substrate, a culture substrate with high cell adhesion and adsorption properties is extremely important.
[0028] 生体内の軟骨組織には血管が発達して 、な 、ことから低酸素条件となって 、ること[0028] Blood vessels develop in cartilage tissue in vivo, which results in hypoxic conditions.
、また、体重による圧負荷を受けていることから、これらの生体条件に近い条件での 培養も有効と考えられる。このため軟骨細胞の培養では、 1〜15%の低酸素条件下 で行うことや、 0. l〜20MPa (周期負荷の場合には 0. 01〜2Hz)の圧をかけて培 養を行うこと、およびこれらの条件を組み合わせて培養することも可能である。圧を負 荷する方法については、具体的にはポンプやピストン状のものを用いて培地に空気 圧や水圧を加える方法等がある。 In addition, since they are subjected to pressure load due to their body weight, culturing under conditions similar to these biological conditions is also considered effective. For this reason, when culturing chondrocytes, it is necessary to perform the culture under hypoxic conditions of 1 to 15% or by applying a pressure of 0.1 to 20 MPa (0.01 to 2 Hz in the case of cyclic loading). , and it is also possible to culture using a combination of these conditions. Specific methods for applying pressure include applying air or water pressure to the culture medium using a pump or piston-like device.
[0029] さらに、培養容器を回転させる培養方式または培地を振とうしながら培養する方法を 行うことにより、生体外での培養によって基材内部まで軟骨組織の主要構成成分で あるタイプ IIコラーゲンカゝら成る移植用軟骨組織が得られる。 [0029] Furthermore, by performing a culture method in which the culture container is rotated or a method in which the culture medium is shaken, type II collagen, which is a main component of cartilage tissue, can be cultured in vitro to the inside of the base material. A cartilage tissue for transplantation consisting of the following is obtained.
[0030] 軟骨細胞の培養では、少なくとも細胞外マトリックスが形成されるまで行う。通常、培 養 2〜4週間程度で軟骨細胞が本発明の培養基材の上に良好に接着、増殖し、コラ ゲン様の細胞外マトリックスが形成される。 [0030]Culturing of chondrocytes is carried out until at least an extracellular matrix is formed. Usually, chondrocytes adhere well to and proliferate on the culture substrate of the present invention after about 2 to 4 weeks of culture, and a collagen-like extracellular matrix is formed.
[0031] このようにして製造される、本発明の、キトサンと酸性生体高分子とのハイブッリド繊 維よりなる培養基材、及び培養基材に付着した軟骨組織を含むものは、軟骨損傷の 修復のための移植用基材として好適に用いることができる。 [0031] The culture substrate of the present invention produced in this way, which is made of a hybrid fiber of chitosan and an acidic biopolymer, and the culture substrate containing cartilage tissue attached to the culture substrate can be used to repair cartilage damage. It can be suitably used as a substrate for transplantation.
以下に実施例によって本発明をさらに説明するが、本発明が実施例に限定される ものではな!/、ことは勿論である。 The present invention will be further explained below with reference to Examples, but it goes without saying that the present invention is not limited to the Examples.
実施例 Example
[0032] ¾細 [0032] ¾Fine
キトサン ヒアルロン酸のハイブリッド繊維の製诰 Production of hybrid fibers of chitosan and hyaluronic acid
3. 5 (重量/容量)％のキトサン (君津化学工業社製、 B、分子量:約 600, 000)を 2 %酢酸水溶液に溶解した溶液をカラム (ガラス製、内径 45mm、長さ 410mm)に詰め 、濾布で加圧 (0. 6kgfZcm2)濾過した。この濾液を紡糸用カラム (ガラス製、内径 4 5mm,長さ 410mm)に詰め、これを紡糸液として簡易紡糸装置を用い、以下のような 方法によって繊維を作製した。 50ホール（小孔： θ. 1mm)のノズルから、 0. 8kgf Zcm2の加圧下で飽和塩ィ匕カルシウム溶液中（第 1凝固浴:水 Zメタノール = 1/1 ( 容量）、浴長 100cm、容量約 2L)に上記紡糸液を押出し、次に水 Zメタノール = 1Z 1 (容量）に浸漬 (第 2凝固浴:浴長 50cm、容量約 1L)し、さらに 0. 05%ヒアルロン酸 溶液 (水 Zメタノール = 1Z1 (容量)）を上流側から滴下した中を通過させた。その後 、ローラー (第丄ローラー:速度 4. 4mZ分、第 2ローラー: 4. 5mZ分、延伸倍率 1. 0 2)にかけ、最後に卷取りローラーで巻き取った後、 0. 2% (重量 Z容量)の水酸化ナ トリウム溶液 (水 Zメタノール = 1Z9 (容量)）に約 15時間浸漬後、水洗し、さらにメタ ノールに約 30分浸漬後取り出しローラーから糸状にほどき、 80°C程度の温風で乾燥 させ、しなやかなキトサン一ヒアルロン酸ノヽイブリツド繊維を得た。 3. A solution of 5 (weight/volume) chitosan (manufactured by Kimitsu Chemical Co., Ltd., B, molecular weight: approx. 600,000) dissolved in 2% acetic acid aqueous solution was added to a column (made of glass, inner diameter 45 mm, length 410 mm). The mixture was packed and filtered using a filter cloth under pressure (0.6 kgfZcm2). This filtrate was transferred to a spinning column (glass, inner diameter 4 5 mm, length 410 mm), and using this as a spinning solution in a simple spinning device, fibers were produced by the following method. From a nozzle with 50 holes (small hole: θ. 1 mm), it was coagulated in a saturated salt-calcium solution under a pressure of 0.8 kgf Zcm2 (first coagulation bath: water Z methanol = 1/1 (volume), bath length 100 cm, The above spinning solution was extruded into a solution (volume: approx. 2 L), then immersed in water (volume) (2nd coagulation bath: bath length: 50 cm, volume: approx. 1 L), and then immersed in 0.05% hyaluronic acid solution (water). Zmethanol = 1Z1 (volume)) was dropped from the upstream side and passed through. After that, it is applied to rollers (first roller: speed 4.4mZ , second roller: 4.5mZ, stretching ratio 1.02), and finally wound up with a winding roller, 0.2% (weight Z After immersing it in a sodium hydroxide solution (water Z methanol = 1Z9 (volume)) for about 15 hours, washing it with water, and immersing it in methanol for about 30 minutes, take it out, unwind it into a thread from the roller, and store it at about 80°C. By drying with warm air, supple chitosan-hyaluronic acid hybrid fibers were obtained.
[0033] 実飾 12 [0033] Mikasa 12
ノ、イブリツド繊維からの谘着某材「ίΊの作製 ノ、Preparation of a certain material ``ίΊ'' from hybrid fibers
実施例 1で製造したハイブリッド繊維を撚糸した後、市販の加工機器を用いて約 40 0 mの糸と糸との間隙を有する織物状構造物を作製した。この織物状構造物を 4枚 重ねて縫合し、培養基材 [1]を作製した（図 1)。この培養基材の空隙率は 66. 0%で めつに。 After twisting the hybrid fibers produced in Example 1, a woven structure having a gap between yarns of about 400 m was produced using commercially available processing equipment. Four layers of this woven structure were stacked and sewn together to create a culture substrate [1] (Figure 1). The porosity of this culture substrate is 66.0%.
[0034] 実飾 13 [0034] Mikasa 13
ノ、イブリツド繊維からの谘着某材「2Ίの作製 ノ、Preparation of a certain material ``2Ί'' from hybrid fibers
実施例 1で製造したハイブリッド繊維は 50本の束であることからこれをほぐして塊状 にし、綿状物を作製した。また、ハイブリッド繊維を撚糸した後、約 400 mの糸と糸 との間隙を有する織物を作製し、 2枚の織物で前記綿状物を挟み込むように成形し、 培養基材 [2]を作製した (図 1)。この培養基材の空隙率は 85. 0%であった。 Since the hybrid fiber produced in Example 1 was a bundle of 50 fibers, it was loosened into a lump to produce a cotton-like material. In addition, after twisting the hybrid fibers, a woven fabric with a gap of about 400 m between the threads was produced, and the cotton-like material was sandwiched between the two woven fabrics to form a culture substrate [2]. (Figure 1). The porosity of this culture substrate was 85.0%.
[0035] 実施例 4 [0035] Example 4
ノ、イブリツド繊維からの焙着某材「3Ίの作製 ノ、Preparation of a certain roasted material ``3Ί'' from hybrid fibers
実施例 1で製造したハイブリッド繊維から微細な綿状構造物を作製した。また、撚糸 した実施例 1で製造したノヽイブリツド繊維力も約 400 μ mの糸と糸との間隙を有する 織物状構造物を作製し、筒状に成形加工した後、この内部に前記綿状構造物を詰 めて培養基材 [3]を作製した（図 1)。この培養基材の空隙率は 92. 6%であった。 A fine cotton-like structure was produced from the hybrid fiber produced in Example 1. In addition, the twisted fibers produced in Example 1 were fabricated into a woven structure having a gap of about 400 μm between the threads, and after being formed into a cylinder, the cotton-like structure was placed inside the fabric. pack things A culture substrate [3] was prepared for the first time (Fig. 1). The porosity of this culture substrate was 92.6%.
[0036] 実施例 5 [0036] Example 5
焙着某材を用いた軟骨細胞の焙着 (1) Roofing of chondrocytes using a certain roasting material (1)
実施例 2に記載の培養基材 [1]を用いて軟骨細胞の培養試験を実施した。 Kawasa kiら、および Yasuiらの方法（Kawasaki, K.等， J.Cell Physiol, 179, 142-148(1999), Ya sui, N.等， Exp.Cell Biol., 50, 92-100(1982))に準じて軟骨細胞の採取および培養を 行った。すなわち、 日本白色家兎 (8週齢、体重 1. 8〜2. Okg)の膝関節部位カも軟 骨組織片を採取し、 0. 25%トリプシン溶液を添加して 37°Cで 25分間処理した後、 0 . 25%コラーゲナーゼ (タイプ II)溶液を添加し、 37°Cで 4時間程度処理を行い、細 胞を単離した。この細胞浮遊液を 50 L採取しトリパンブルー 50 Lをカ卩え、よく撹 拌した後、 20 Lを血球計算盤に乗せて細胞数をカウントし、全細胞数を算出した。 予めオートクレーブで滅菌処理を行った培養基材をマルチウヱルプレート（12ゥエル 、 Falcon社製）に入れ、基材上に各基材当たり 3 X 105個となるように軟骨細胞浮遊 液 80 Lを添カ卩した。 5%CO存在下、 37°Cの培養器で 1時間インキュベートした後 A chondrocyte culture test was conducted using the culture substrate [1] described in Example 2. Methods of Kawasaki et al. and Yasui et al. )) Chondrocytes were collected and cultured. Specifically, pieces of cartilage tissue were collected from the knee joint of a Japanese white rabbit (8 weeks old, weight 1.8 to 2.0 kg), and a 0.25% trypsin solution was added and incubated at 37°C for 25 minutes. After the treatment, a 0.25% collagenase (type II) solution was added, and treatment was performed at 37°C for about 4 hours to isolate the cells. 50 L of this cell suspension was collected, 50 L of trypan blue was added thereto, and after stirring well, 20 L was placed on a hemocytometer and the number of cells was counted to calculate the total number of cells. Place culture substrates that have been sterilized in an autoclave in advance into a multi-well plate (12 wells, manufactured by Falcon), and add 80 L of chondrocyte suspension on each substrate at a density of 3 x 10 5 cells per substrate. I added the following. After 1 hour incubation at 37°C in 5% CO
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、 DMEM培地 2mLを少量ずつ添カ卩し、さらに 0. 1%ァスコルビン酸ホスフェート 20 μ Lをカ卩えて、上記条件下で 8週間培養を行った。 比較対照として、織物状構造物の糸と糸との間隙が約 200 mの培養基材につい ても同様に培養試験を実施した。なお、タンパク量は Lowry法に、また、酸性ムコ多糖 量はジメチルメチレンブルーを用いた Farndaleらの方法（Farndale, R.W.,等 Biochimi ca et Biophysica Acta, 883, 173-177 (1986))に準じて測定した。 Then, 2 mL of DMEM medium was added in small portions, and 20 μL of 0.1% ascorbic acid phosphate was added thereto, followed by culturing under the above conditions for 8 weeks. As a comparison, a culture test was also conducted on a culture substrate in which the gap between the threads of a woven structure was approximately 200 m. The protein content was measured according to the Lowry method, and the acidic mucopolysaccharide content was measured according to the method of Farndale et al. using dimethylmethylene blue (Farndale, R.W., et al. Biochimi ca et Biophysica Acta, 883, 173-177 (1986)). did.
[0037] 図 2には 21日間培養した際のタンパク質量の変化を示した力 実施例 2に記載の 糸と糸との間隙が 400 mの培養基材を用いた場合、比較対照とした間隙 200 m の培養基材 (空隙率 59. 3%)と比較してタンパク質量の増加が良好であった。また、 図 3には酸性ムコ多糖量の変化を示した力 間隙 400 mの培養基材では培養に伴 つて顕著に増加した。これに対し間隙が 200 mの培養基材では酸性ムコ多糖の産 生はほとんど見られな力つた。 [0037] Figure 2 shows the change in protein amount when cultured for 21 days. The increase in protein content was better compared to the 200 m culture substrate (porosity 59.3%). Furthermore, Figure 3 shows the change in the amount of acidic mucopolysaccharide, which significantly increased with culture in the culture substrate with a gap of 400 m. In contrast, in the culture substrate with a gap of 200 m, almost no acidic mucopolysaccharide production was observed.
[0038] 実施例 6 焙着某材を用いた軟骨細胞の焙着 (2) [0038] Example 6 Roofing of chondrocytes using a certain roasting material (2)
実施例 3の培養基材 [2]を用いて実施例 5と同様な方法により軟骨細胞の培養試 験を実施した。比較対照には実施例 2に記載の培養基材 [1]を使用した。図 4にはタ ンパク量の変化を、図 5には酸性ムコ多糖量の変化を示した力 実施例 2に記載した 糸と糸との間隙が 400 /z mの織物状構造物の内部に綿状繊維を含む構造の培養基 材を用いた場合、糸と糸との間隙 400 mの織物状構造物を重ね合わせた構造の 培養基材と比較して、どちらの値とも良好に増加し、特に酸性ムコ多糖の増加が顕著 であった。 A chondrocyte culture test was conducted using the culture substrate [2] of Example 3 in the same manner as in Example 5. The culture substrate [1] described in Example 2 was used for comparison. Figure 4 shows the change in the amount of protein, and Figure 5 shows the change in the amount of acidic mucopolysaccharide. When using a culture substrate with a structure containing fiber-like fibers, both values increased favorably compared to a culture substrate with a structure in which woven structures with a gap of 400 m between the threads were stacked. The increase in acidic mucopolysaccharide was remarkable.
[0039] 実施例 7 [0039] Example 7
谘着某材 用いた軟骨細qの谘着 (3) A piece of cartilage made from a certain material (3)
培養基材 [1]および [2]を用いて、回転培養 (容器ごと回転させて培地に流れをつ くる）方式での軟骨細胞の培養試験を実施した。 Using culture substrates [1] and [2], we conducted a culture test of chondrocytes using a rotation culture method (rotating the entire container to create a flow in the medium).
実施例 5に記載した方法と同様にして軟骨細胞を採集し、マルチウエルプレートに 入れた培養基材に播種した。 5%CO存在下、 37°Cの培養器で 1時間インキュベー Chondrocytes were collected in the same manner as described in Example 5 and seeded on culture substrates placed in multi-well plates. Incubate for 1 hour in an incubator at 37°C in the presence of 5% CO.
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トした後、 DMEM培地 2mLを少量ずつ添カ卩し、さらに 0. 1%ァスコルビン酸ホスフエ ート 20 Lを加えて上記条件下で 1週間静置培養を行った。この培養基材を専用の 培養容器に入れ、市販の装置 (Synthecon社製)を用いて 5%CO存在下、 37°Cで 7 After that, 2 mL of DMEM medium was added little by little, and 20 L of 0.1% ascorbic acid phosphate was added, followed by static culture for one week under the above conditions. This culture substrate was placed in a special culture container and incubated at 37°C in the presence of 5% CO using a commercially available device (manufactured by Synthecon).
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週間、回転培養を行った (合計 8週間）。なお、 DNA量は発色試薬である Hoechst33 258を用いて測定した。 Rotary culture was performed for 1 week (8 weeks in total). Note that the amount of DNA was measured using Hoechst33 258, a coloring reagent.
[0040] 図 6には培養組織の湿重量、タンパク量および DNA量の変化を示した。培養基材 [0040] Figure 6 shows changes in wet weight, protein content, and DNA content of cultured tissues. Culture substrate
[1]および [2]のどちらを用いた場合でも全ての値が培養に伴って良好に増加した 1S 両基材の差は見られな力つた。一方、図 9には 5週間および 8週間培養した軟骨 組織のタイプ IIコラーゲンの抗体染色像を示したが、培養基材 [2]を用いた場合には 、基材内部までタイプ Πコラーゲンが十分形成されていた。これに対し、培養基材 [1] では内部でのタイプ IIコラーゲンの産生が不十分であった。 Regardless of whether [1] or [2] was used, all values increased favorably with 1S culture. No differences were observed between the two substrates. On the other hand, Figure 9 shows antibody-stained images of type II collagen in cartilage tissue cultured for 5 and 8 weeks, but when culture substrate [2] was used, type Π collagen was sufficiently absorbed into the interior of the substrate. was being formed. In contrast, type II collagen production within the culture substrate [1] was insufficient.
[0041] 静置培養を行った場合には、正常軟骨組織には含まれていないタイプ Iコラーゲン の産生が若干見られたが、回転培養を行うことによって、どちらの基材を使用した場 合でもタイプ Iコラーゲンの産生は全く見られな力つた。 [0042] なお、生分解性の合成繊維であるポリダラクチン繊維を用いて培養基材 [1]と同様 の基材を作成し、比較対象として同様の培養試験を行ったところ、 3週間の培養でポ リグラクチン繊維は溶解し、基材形状の維持は困難であった。 [0041] When static culture was performed, some production of type I collagen, which is not included in normal cartilage tissue, was observed, but by performing rotational culture, it was possible to see the production of type I collagen, which is not included in normal cartilage tissue. However, no type I collagen production was observed. [0042] Furthermore, when a culture substrate similar to [1] was prepared using polydlactin fiber, which is a biodegradable synthetic fiber, and a similar culture test was conducted for comparison, it was found that after 3 weeks of culture, The polyglactin fibers dissolved and it was difficult to maintain the shape of the base material.
[0043] 実施例 8 [0043] Example 8
焙着某材を用いた軟骨細胞の焙着 (4) Roofing of chondrocytes using a certain roasting material (4)
培養基材 [ 1 ]および [ 2]を用いて、回転培養と低酸素条件を併用した培養方式で の軟骨細胞の培養試験を実施した。 Using culture substrates [1] and [2], we conducted a culture test of chondrocytes using a culture method that combined rotation culture and hypoxic conditions.
実施例 7に記載した方法と同様にして 1週間の静置培養を行った後、専用の培養 容器に入れ、 5%CO存在下、 37°C、通常の酸素濃度下（20%O )で回転培養を 1 After statically culturing for one week in the same manner as described in Example 7, the cells were placed in a special culture container and incubated at 37°C in the presence of 5% CO and under normal oxygen concentration (20% O ). Rotating culture 1
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週間行った。次いで、酸素濃度を 5%に設定した培養器中（5%CO、 37°C)で 6週 I went for a week. Then, for 6 weeks in an incubator with an oxygen concentration of 5% (5% CO, 37°C).
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間、回転培養を行った (合計 8週間)。なお、培養組織の硬度は万能試験機 (AGS— H、島津製作所製)を用いて測定した圧縮強度曲線力 算出したヤング率で表記し た。 Rotary culture was performed for a period of 8 weeks (total of 8 weeks). The hardness of the cultured tissue was expressed as the Young's modulus calculated from the compressive strength curve force measured using a universal testing machine (AGS-H, manufactured by Shimadzu Corporation).
[0044] 図 8には培養組織の湿重量、タンパク量および DNA量の変化を示した。実施例 5と 同様、両基材とも培養に伴って全ての値が通常の 20%O下での回転培養（図 5)に [0044] Figure 8 shows changes in wet weight, protein content, and DNA content of cultured tissues. As in Example 5, all values for both substrates changed as they were cultured under normal rotational culture under 20% O (Figure 5).
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比べて顕著に増加したが、基材構造による差は見られな力つた。一方、図 13にはタ イブ IIコラーゲンの抗体染色像を示したが、培養基材 [1]を用いた場合には 5週間の 培養では基材中央部分におけるタイプ IIコラーゲンの産生が不十分であつたのに対 し、培養基材 [2]を用いた場合には、 5週間の培養で基材内部まで良好にタイプ IIコ ラーゲンが産生されていた。また、図 14には培養基材 [2]を用いて培養した組織の 硬度変化を示したが、培養 5週目以降に急激な硬度の上昇が見られた。これは、タイ プ IIコラーゲンの産生によって正常軟骨組織と同様の硬い軟骨組織が形成されたこ とによるものと考えられる。これらのことから、培養基材 [2]を用い、回転培養と低酸素 条件を組み合わせることによって、通常の 20%O下より短期間の 5週間の培養によ However, no difference was observed depending on the structure of the base material. On the other hand, Figure 13 shows an antibody-stained image of type II collagen, which shows that when culture substrate [1] was used, the production of type II collagen in the center of the substrate was insufficient after 5 weeks of culture. In contrast, when culture substrate [2] was used, type II collagen was well produced inside the substrate after 5 weeks of culture. Furthermore, Figure 14 shows changes in the hardness of tissues cultured using the culture substrate [2], and a rapid increase in hardness was observed after the 5th week of culture. This is thought to be due to the formation of hard cartilage tissue similar to normal cartilage tissue due to the production of type II collagen. For these reasons, by using a culture substrate [2] and combining rotational culture and low oxygen conditions, it was possible to culture for 5 weeks, which is shorter than the usual 20% O2 environment.
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つても軟骨組織が十分形成され、且つ、基材内部まで十分、タイプ IIコラーゲンで占 められた良好な移植用軟骨組織を得ることができる。 It is possible to obtain a good cartilage tissue for transplantation in which the cartilage tissue is sufficiently formed and the interior of the base material is sufficiently occupied by type II collagen.
[0045] 実施例 9 [0045] Example 9
焙着某材を用いた軟骨細胞の焙着 (5) 実施例 4に記載の培養基材 [3]を用いて、回転培養方式での軟骨細胞の培養試 験を実施した。実施例 7に記載した方法と同様にして 1週間の静置培養を行った後、 専用の培養容器に入れ、 5%COおよび 20%O下、 37°Cで 7週間、回転培養を行 Roofing of chondrocytes using a certain roasting material (5) Using the culture substrate [3] described in Example 4, a chondrocyte culture test was conducted using a rotational culture method. After statically culturing for 1 week in the same manner as described in Example 7, the cells were placed in a special culture container and rotary cultured for 7 weeks at 37°C under 5% CO and 20% O.
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つた (合計 8週間)。 ivy (8 weeks total).
[0046] 図 11には培養組織の湿重量、タンパク量および DNA量の変化を示した。培養基 材 [2]および [3]の両基材とも培養に伴って全ての値が増加したが、基材構造による 明確な差は見られな力つた。一方、図 18には培養基材 [3]を用いた場合のタイプ IIコ ラーゲンの抗体染色像を示したが、 5週間の培養ではやや空隙が見られるものの、 8 週間培養を行うことによって内部まで良好にタイプ IIコラーゲンが産生しているのを確 認した。また、培養基材 [2]を用いて培養した場合には、基材を取り囲むように組織 が形成されるため、内部のみを取り出して移植することは困難であるが、培養基材 [3 ]を用いて培養を行った場合には、表面の織物状構造物を取り除いて、内部のタイプ Πコラーゲンで満たされた綿状構造物部分のみを移植することも可能であった。 [0046] Figure 11 shows changes in wet weight, protein content, and DNA content of cultured tissues. For both culture substrates [2] and [3], all values increased with culture, but no clear differences were observed depending on the structure of the substrate. On the other hand, Figure 18 shows an antibody-stained image of type II collagen when culture substrate [3] was used. Although some voids were observed after 5 weeks of culture, internal areas were cleared after 8 weeks of culture. It was confirmed that type II collagen was being produced well up to the point where it was removed. Furthermore, when cultured using a culture substrate [2], tissue is formed to surround the substrate, making it difficult to remove only the inside and transplant it. When culturing was carried out using the collagen, it was possible to remove the surface fabric-like structure and transplant only the inner flocculent structure filled with type Π collagen.
[0047] 実施例 10 [0047] Example 10
谘着某材 用いた 細胞の谘着 A collection of cells using a certain material
実施例 3に記載の基材 [2]を用いて、幹細胞の培養試験を実施した。 Wakitani等 の方法（Wakitani, S.等， J. Bone Joint Surg. Am., 76, 579- 592(1994))に準じて、日 本白色家兎 (8週齢)の顎骨より骨髄液を採取し、 10%の FBSおよび 1%の抗生物質 (ペニシリンとストレプトマイシンとファンギゾンの混液）を含む DMEM培地中で 14日 間培養を行った。この後、培養容器底面の接着細胞 (骨髄間質細胞および骨髄間葉 系細胞）のみを回収し、さらに培養を行った。 A stem cell culture test was conducted using the substrate [2] described in Example 3. Bone marrow fluid was collected from the jawbone of a Japanese white rabbit (8 weeks old) according to the method of Wakitani et al. The cells were then cultured for 14 days in DMEM medium containing 10% FBS and 1% antibiotics (mixture of penicillin, streptomycin, and fungizone). After this, only the adherent cells (bone marrow stromal cells and bone marrow mesenchymal cells) on the bottom of the culture vessel were collected and further cultured.
予めオートクレーブで滅菌処理を行った培養基材をマルチウエルプレート（24ゥェ ル）に入れ、基材当たり 2. 5xl07個となるように増殖させた骨髄細胞を含む細胞浮 遊液 25 1を播種した。 5%CO存在下、 37°Cの培養器で 1時間インキュベートした Place culture substrates that have been sterilized in an autoclave in advance into a multi-well plate (24 wells), and add a cell suspension containing 2.5 x 10 bone marrow cells grown to 7 cells per substrate. Sowed. Incubated for 1 hour in an incubator at 37°C in the presence of 5% CO.
2 2
後、軟骨誘導用培地 lmlを少量ずつ添加し、上記条件下で 21日間培養を行った。 なお軟骨誘導用培地には、抗性物質 (ペニシリンとストレプトマイシンとファンギゾンの 混液） 1%、デキサメサゾン 0. l ^ M,ピルビン酸 100 μ g/mUァスコルビン酸— 2 —ホスフェート 50 μ g/m プロリン 40 μ gZmlおよび TGF— β 10ng/mlを含む DMEM— high glucose培地を使用した。 Thereafter, 1 ml of cartilage induction medium was added little by little, and cultured under the above conditions for 21 days. The cartilage induction medium contained 1% antibiotics (mixture of penicillin, streptomycin, and fungizone), dexamethasone 0. l ^ M, pyruvate 100 μg/mU, ascorbic acid 2 —phosphate 50 μg/m proline 40 μgZml and TGF—contains β 10ng/ml DMEM-high glucose medium was used.
図 19には 21日間培養した際の組織のタイプ IIコラーゲンの抗体染色像を示したが 、基材内部まで十分タイプ IIコラーゲンが形成されており、本基材により骨髄幹細胞 力も軟骨細胞への分ィ匕が良好に起こっていることを確認した。 Figure 19 shows an antibody-stained image of type II collagen in the tissue after 21 days of culture, and it shows that type II collagen has been sufficiently formed inside the base material, and this base material also allows the distribution of bone marrow stem cell power to chondrocytes. It was confirmed that the phishing was occurring properly.
[0048] 比較例 2 [0048] Comparative example 2
ノ、イブリツド繊維からの焙着某材の作製 2. Production of roasted material from hybrid fibers
実施例 1で製造したハイブリッド繊維から微細な綿状構造物を作製した。また、撚糸 した実施例 1で製造したノヽイブリツド繊維力も約 600 μ mの糸と糸との間隙を有する 織物状構造物を作製し、筒状に成形加工した後、この内部に前記綿状構造物を詰 めて培養基材 [4]を作製した。この培養基材の空隙率は 95. 8%であった。 A fine cotton-like structure was produced from the hybrid fiber produced in Example 1. In addition, the twisted fibers produced in Example 1 were fabricated into a woven structure having a gap of about 600 μm between the threads, formed into a cylindrical shape, and then the cotton-like structure was inserted into the inside of the woven structure. A culture substrate [4] was prepared by filling it with material. The porosity of this culture substrate was 95.8%.
[0049] 実施例 11 [0049] Example 11
谘着某材 用いた軟骨細胞の谘着 Selected material: Selected chondrocytes used
比較例 2の培養基材 [4]を用いて、回転培養方式での軟骨細胞の培養試験を実施 した。比較対照には実施例 4に記載の培養基材 [3]を使用した。実施例 7に記載した 方法と同様にして 1週間の静置培養を行った後、専用の培養容器に入れ、 5%CO Using the culture substrate [4] of Comparative Example 2, a chondrocyte culture test was conducted using a rotational culture method. The culture substrate [3] described in Example 4 was used for comparison. After statically culturing for one week in the same manner as described in Example 7, the cells were placed in a special culture container and incubated with 5% CO2.
2 および 20%0下、 37°Cで 7週間、回転培養を行った (合計 8週間）。 2 and 20% 0 at 37°C for 7 weeks (total 8 weeks).
2 2
空隙率 92. 6%の実培養基材 [3]を用いて培養した場合には、タンパク量および D NA量は図 16および図 17とほぼ同様に両者とも良好に増加した力 空隙率 95. 8% の培養基材 [4]を用いて培養した場合には、このような良好な増加は見られなカゝつた 。また、タイプ IIコラーゲンの抗体染色を行った結果、タイプ IIコラーゲンの産生も図 1 8に比べて不十分であった。 Porosity 92. When cultured using a 6% actual culture substrate [3], the protein content and DNA content both increased favorably as shown in Figures 16 and 17. Such a good increase was not observed when cultured using 8% culture substrate [4]. Furthermore, as a result of antibody staining of type II collagen, production of type II collagen was also insufficient compared to Figure 18.
産業上の利用可能性 Industrial applicability
[0050] 再生医療のための培養基材として利用できる。 [0050] It can be used as a culture substrate for regenerative medicine.

Claims

請求の範囲 The scope of the claims
[1] 繊維内部がキトサンまたはその塩よりなり、繊維表面がキトサンとヒアルロン酸、コン ドロイチン硫酸、およびデルマタン硫酸よりなる群から選択される酸性生体高分子と の複合体で被覆されているキトサン Z酸性生体高分子ハイブリッド繊維よりなる織物 または編物を含む軟骨組織再生用培養基材であって、織物または編物の糸と糸の 間隙は 250〜500 μ mであり、基材内部の空隙率が 65〜94%である培養基材。 [1] Chitosan Z, in which the inside of the fiber is made of chitosan or its salt, and the fiber surface is coated with a complex of chitosan and an acidic biopolymer selected from the group consisting of hyaluronic acid, chondroitin sulfate, and dermatan sulfate. A culture substrate for cartilage tissue regeneration that includes a woven or knitted fabric made of acidic biopolymer hybrid fibers, in which the gap between the threads of the woven or knitted fabric is 250 to 500 μm, and the porosity inside the substrate is 65 Culture substrate that is ~94%.
[2] 織物または編物を重ねてなる請求項 1に記載の軟骨組織再生用培養基材。 [2] The culture substrate for cartilage tissue regeneration according to claim 1, which is formed by stacking woven or knitted fabrics.
[3] 織物または編物の間に、請求項 1に記載のキトサン Z酸性生体高分子ハイブリッド 繊維よりなる綿状物が挟まれて!/ヽる請求項 1に記載の軟骨組織再生用培養基材。 [3] The culture substrate for cartilage tissue regeneration according to claim 1, wherein a cotton-like material made of the chitosan Z acidic biopolymer hybrid fiber according to claim 1 is sandwiched between the woven or knitted fabrics. .
[4] 織物または編物で作った円筒中に、請求項 1に記載のキトサン Z酸性生体高分子 ハイブリッド繊維よりなる綿状物が充填されて 、る請求項 1に記載の軟骨組織再生用 培養基材。 [4] The culture medium for cartilage tissue regeneration according to claim 1, wherein a cylinder made of a woven or knitted fabric is filled with a cotton-like material made of the chitosan Z acidic biopolymer hybrid fiber according to claim 1. Material.
[5] 請求項 1〜4の!ヽずれかに記載の培養基材を用いて生体外で軟骨細胞を培養する 方法。 [5] A method for culturing chondrocytes in vitro using the culture substrate according to any one of claims 1 to 4.
[6] 培養容器を回転させながら培養を行う請求項 5に記載の培養方法。 [6] The culture method according to claim 5, wherein the culture is performed while rotating the culture container.
[7] 培地を振とうしながら培養する請求項 5に記載の培養方法。 [7] The culture method according to claim 5, wherein the culture medium is cultured while shaking.
[8] 低酸素条件下で培養する請求項 5〜7の 、ずれかに記載の培養方法。 [8] The culture method according to any one of claims 5 to 7, wherein the culture is carried out under hypoxic conditions.
[9] 請求項 5〜8の!ヽずれかに記載の培養方法で得られる移植用軟骨組織。 [9] A cartilage tissue for transplantation obtained by the culture method according to any one of claims 5 to 8.
PCT/JP2006/324241 2005-12-06 2006-12-05 Culture substrate and culture method for cartilage tissue regeneration WO2007066650A1 (en)

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