JP2012000262A - Human chondrocyte and method of producing cartilage issue with novel support - Google Patents
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本発明は、軟骨組織の製法に関し、より詳細には、軟骨幹細胞と担体材料とを用いて組織工学的手法により調製された再生軟骨組織の製造方法に関する。 The present invention relates to a method for producing a cartilage tissue, and more particularly to a method for producing a regenerated cartilage tissue prepared by a tissue engineering technique using a cartilage stem cell and a carrier material.
組織工学的な手法によりヒト軟骨組織を作製するためには、軟骨細胞・軟骨幹/前駆細胞と支持体としての３次元的な多孔質性の足場材料（担体）が必要である。 In order to produce human cartilage tissue by a tissue engineering technique, chondrocytes / cartilage stem / progenitor cells and a three-dimensional porous scaffold material (carrier) as a support are required.
このような足場材料は多孔質性や生体親和性や生体吸収性などの条件が要求され、従来、コラーゲンなどの天然高分子で調製した３次元的な多孔質性材料 あるいはポリ乳酸（ＰＬＡ）やポリグリコール酸（ＰＧＡ）や乳酸とグリコール酸との共重合体（ＰＬＧＡ）のような生体吸収性合成高分子などが用いられている（特許文献１〜５、非特許文献１〜５）。 Such scaffold materials are required to have conditions such as porosity, biocompatibility, and bioabsorbability. Conventionally, three-dimensional porous materials prepared from natural polymers such as collagen, polylactic acid (PLA), Bioabsorbable synthetic polymers such as polyglycolic acid (PGA) and a copolymer of lactic acid and glycolic acid (PLGA) are used (Patent Documents 1 to 5, Non-Patent Documents 1 to 5).
生体外で細胞から三次元軟骨組織構築を行う場合、通常適当な足場材料を用いて３次元培養を行うか、攪拌培養を行っていた（特許文献６、非特許文献６）。しかし、細胞に与えられる機械的刺激や損傷が強く、大きな組織を得ることは困難であった。 When constructing a three-dimensional cartilage tissue from cells ex vivo, usually three-dimensional culture is performed using a suitable scaffold material or stirring culture is performed (Patent Document 6, Non-Patent Document 6). However, mechanical stimulation and damage given to cells are strong, and it has been difficult to obtain large tissues.
近年、ＮＡＳＡが開発したガス交換機能を備えた回転式ＲＷＶ（Ｒｏｔａｔｉｎｇ−Ｗａｌｌ Ｖｅｓｓｅｌ）バイオリアクターによる培養法が開発された。バイオリアクター内は、回転による応力のため、地上の重力に比較して１００分の１程度の微小重力環境となり、細胞は培養液中に均一に懸濁された状態で浮遊しており、増殖・凝集して、大きな組織塊を形成することが可能となった（特許文献７、８、非特許文献７、８、９）。 In recent years, a culture method using a rotating RWV (Rotating-Wall Vessel) bioreactor with a gas exchange function developed by NASA has been developed. The bioreactor has a microgravity environment that is about 1 / 100th of the ground due to stress due to rotation, and the cells are suspended in a uniform suspension in the culture medium. Aggregates to form a large tissue mass (Patent Documents 7 and 8, Non-Patent Documents 7, 8, and 9).
生体由来の天然高分子であるコラーゲンは、親水性で、細胞との相互作用が非常に優れており、細胞の播種の容易なものであるが、機械強度が低く、柔らかくてくずれやすい。一方、生体吸収性合成高分子であるPLAやPGAは、機械強度に優れているものの、疎水性であり、その大きい隙間のために大部分の細胞は隙間を通過してしまい、細胞を播種することが極めて困難である。さらに、生体内で吸収される際の炎症により細胞や組織が傷害されるために、その後の形態を維持できない。このように臨床応用に耐え得るヒト軟骨組織の作製のための多孔質性足場材料はまだ開発されていない。 Collagen, which is a natural polymer derived from a living body, is hydrophilic and has an excellent interaction with cells and is easy to seed cells, but has low mechanical strength and is soft and easily broken. On the other hand, PLA and PGA, which are bioabsorbable synthetic polymers, are excellent in mechanical strength but are hydrophobic, and because of the large gap, most cells pass through the gap and seed the cells. Is extremely difficult. Furthermore, since the cells and tissues are damaged by inflammation when absorbed in vivo, the subsequent form cannot be maintained. Thus, a porous scaffold material for producing a human cartilage tissue that can withstand clinical application has not been developed yet.
擬微小重力環境下での細胞培養において、どのような足場材料が好適であるかは従来は不明であった。すなわち、三次元培養の足場材料として一般的なコラーゲンスポンジ等は機械的に充分な強度を持たず、それらを用いたヒト軟骨組織は力学的な物性が劣っているという欠点があった。すなわち、従来技術では臨床応用に耐えられるようなヒト軟骨組織を作製することができなかった。 In the past, it was unknown what kind of scaffold material is suitable for cell culture in a pseudo-microgravity environment. That is, collagen sponges or the like that are common as scaffold materials for three-dimensional culture do not have sufficient mechanical strength, and human cartilage tissues using them have the disadvantage of poor mechanical properties. That is, the conventional art cannot produce human cartilage tissue that can withstand clinical application.
本発明は、新しいヒト軟骨組織の作製方法を実現する新規技術を提供することを目的とする。 An object of this invention is to provide the novel technique which implement | achieves the preparation method of a new human cartilage tissue.
本発明は、上記のような多孔質性担体材料の従来の問題点を解決するために、新規多孔質性担体（ハイドロキシアパタイト/コンドロイチン硫酸-コラーゲン複合体：HAp/ChS-Colあるいはハイドロキシアパタイト-コラーゲン複合体：HAp -Col）とヒト軟骨細胞あるいはヒト軟骨幹/前駆細胞を組み合わせることによって、臨床応用の可能な新しいヒト軟骨組織の作製方法を実現する新規技術である。 The present invention provides a novel porous carrier (hydroxyapatite / chondroitin sulfate-collagen complex: HAp / ChS-Col or hydroxyapatite-collagen in order to solve the conventional problems of the porous carrier material as described above. This is a new technology that realizes a new human cartilage tissue preparation method that can be applied clinically by combining the complex: HAp-Col) with human chondrocytes or human cartilage stem / progenitor cells.
本発明では、擬微小重力環境下（浮遊状態）での細胞培養の要素である細胞と足場となる担体材料を新規のものとしている。すなわち、細胞に関しては、ヒト軟骨細胞あるいはヒト軟骨幹/前駆細胞を用いる。また、新規多孔質性担体（ハイドロキシアパタイト/コンドロイチン硫酸-コラーゲン複合体：HAp /ChS-Colあるいはハイドロキシアパタイト-コラーゲン複合体：HAp -Col）は、良好な生体親和性を有し、軟骨細胞等の播種効率が良好で、かつ、ヒト軟骨組織の再構築効率が良好である。さらに、この新規多孔質性担体はハイドロキシアパタイトのナノ粒子を含んでいるために、適度な弾性力を有し機械的強度に優れたヒト軟骨組織を作製できることが大きな特徴である。 In the present invention, a carrier material serving as a scaffold and cells that are elements of cell culture in a pseudo-microgravity environment (floating state) is novel. That is, for the cells, human chondrocytes or human cartilage stem / progenitor cells are used. In addition, the novel porous carrier (hydroxyapatite / chondroitin sulfate-collagen complex: HAp / ChS-Col or hydroxyapatite-collagen complex: HAp-Col) has good biocompatibility, such as chondrocytes The seeding efficiency is good and the remodeling efficiency of human cartilage tissue is good. Furthermore, since this novel porous carrier contains hydroxyapatite nanoparticles, it is a major feature that a human cartilage tissue having an appropriate elasticity and excellent mechanical strength can be produced.
本発明の要旨は以下の通りである。
（１）ヒドロキシアパタイトとコラーゲンを含む多孔質足場材料とともにヒト軟骨膜細胞を生体外で培養することを含む、軟骨再生方法。
（２）多孔質足場材料がさらに多糖を含む（１）記載の方法。
（３）多糖がコンドロイチン硫酸である（２）記載の方法。
（４）ヒト軟骨膜細胞がCD44+CD90+の表現型を有する（１）〜（３）のいずれかに記載の方法。
（５）培養が三次元擬微小重力培養である（１）〜（４）のいずれかに記載の方法。
（６）ヒドロキシアパタイトとコラーゲンを含む多孔質足場材料及びヒト軟骨膜細胞を含む、軟骨再生のための組成物。
The gist of the present invention is as follows.
(1) A method for regenerating cartilage, comprising culturing human perichondrial cells in vitro together with a porous scaffold material containing hydroxyapatite and collagen.
(2) The method according to (1), wherein the porous scaffold material further contains a polysaccharide.
(3) The method according to (2), wherein the polysaccharide is chondroitin sulfate.
(4) The method according to any one of (1) to (3), wherein the human perichondrial cells have a CD44 + CD90 + phenotype.
(5) The method according to any one of (1) to (4), wherein the culture is a three-dimensional pseudo-microgravity culture.
(6) A composition for cartilage regeneration comprising a porous scaffold material containing hydroxyapatite and collagen and human perichondrial cells.
新規多孔質性担体は、構成成分にハイドロキシアパタイトのナノ粒子を含んでいるために、適度な弾性力を有し、機械的強度に優れている。また、良好な生体親和性を有し、軟骨細胞等の播種効率が良く、ヒト軟骨組織の再構築効率が良好である。 Since the novel porous carrier contains hydroxyapatite nanoparticles as constituent components, it has an appropriate elastic force and excellent mechanical strength. In addition, it has good biocompatibility, has good seeding efficiency of chondrocytes, etc., and has good reconstruction efficiency of human cartilage tissue.
細胞に関しても終末分化した成熟ヒト軟骨細胞を使用する方法とともに、高い増殖能・自己複製能・組織再構築能を兼ね備えたヒト軟骨幹/前駆細胞を用いることで、より高品質なヒト軟骨組織の作製が可能となる。 In addition to the method of using mature human chondrocytes with terminal differentiation, human cartilage stem / progenitor cells that have high proliferation ability, self-replication ability, and tissue remodeling ability can be used to improve the quality of human cartilage tissue. Fabrication is possible.
生体外（例えば細胞培養系）において、臨床応用に耐えられるヒト軟骨組織を作製する技術は未開発である。本発明は、生体外の三次元培養系で弾性力と機械的強度を持ち長期的に形態維持されるヒト軟骨組織を作製することができる。 A technique for producing human cartilage tissue that can withstand clinical application in vitro (eg, cell culture system) has not been developed yet. The present invention can produce a human cartilage tissue that has elastic force and mechanical strength and maintains its shape for a long period of time in an in vitro three-dimensional culture system.
以下、本発明の実施の形態についてより詳細に説明する。 Hereinafter, embodiments of the present invention will be described in more detail.
本発明は、ヒドロキシアパタイトとコラーゲンを含む多孔質足場材料とともにヒト軟骨膜細胞を生体外で培養することを含む、軟骨再生方法を提供する。 The present invention provides a method for cartilage regeneration comprising culturing human perichondrial cells in vitro with a porous scaffold material comprising hydroxyapatite and collagen.
多孔質足場材料はさらに多糖を含んでもよく、多糖としては、コンドロイチン硫酸を例示することができる。多孔質足場材料としては、例えば、Adegawa, T.,et al. Preparation and Characterization of Porous Scaffolds Consisted of Hydroxyapatite, Polysaccharides and Collagen for Cartilage Tissue
Engineering Key Eng Mater Vol 396-398, 707-710 (2009)に記載のものを挙げることができる。この文献では、ヒドロキシアパタイト(HＡp)及びコラーゲンを含む多孔質足場材料、並びにヒドロキシアパタイト(HＡp)、コンドロイチン硫酸(ChS)及びコラーゲンを含む多孔質足場材料が凍結乾燥法により作製されている。これらの多孔質足場材料は、HＡp粒子又はHAp /ChS複合体(composite)粒子がコラーゲンマトリックスに含浸されているものである。これらの多孔質足場材料の孔径は直径80-200μmが適当であり、孔は互いに連続している(interconnected)とよい。また、HＡp粒子の平均直径は、1〜20μmが適当であり、5〜10μmが好ましく、例えば、9.3μm とする。HAp/ChS粒子の平均直径は、1〜20μmが適当であり、5〜10μmが好ましく、例えば、8.6μmとする。HApとコラーゲンの質量比は、0.1〜2が適当であり、0.5〜1.5が好ましく、例えば、1：1とする。HAp/Chs中のChsの量は、1〜30質量%が適当であり、1〜10質量%が好ましく、例えば、5.2質量%とする。
The porous scaffold material may further contain a polysaccharide, and examples of the polysaccharide include chondroitin sulfate. Examples of porous scaffold materials include Adegawa, T., et al. Preparation and Characterization of Porous Scaffolds Consisted of Hydroxyapatite, Polysaccharides and Collagen for Cartilage Tissue
Mention may be made of Engineering Key Eng Mater Vol 396-398, 707-710 (2009). In this document, a porous scaffold material containing hydroxyapatite (HAp) and collagen, and a porous scaffold material containing hydroxyapatite (HAp), chondroitin sulfate (ChS) and collagen are produced by a freeze-drying method. These porous scaffold materials are those in which a collagen matrix is impregnated with HAp particles or HAp / ChS composite particles. The pore size of these porous scaffold materials is suitably 80-200 μm in diameter, and the pores should be interconnected. The average diameter of the HAp particles is suitably 1 to 20 μm, preferably 5 to 10 μm, for example, 9.3 μm. The average diameter of the HAp / ChS particles is suitably 1 to 20 μm, preferably 5 to 10 μm, for example, 8.6 μm. The mass ratio of HAp to collagen is suitably 0.1-2, preferably 0.5-1.5, for example 1: 1. The amount of Chs in HAp / Chs is suitably 1 to 30% by mass, preferably 1 to 10% by mass, for example, 5.2% by mass.
以下に多孔質足場材料の作製方法の一例を記載する。HAp及びHAp/ChS複合体を沈殿法で合成する（T.Ikoma et al.: Key Eng. Mater. Vol. 288-289, (2005),pp.159）。純CaCO3粉末を1050℃で3時間か焼する。得られたCaO粉末を蒸留水で300℃にて水和し、Ca(OH)2を生成する。ChS（生化学工業、ウシ気管由来）の添加有り、あるいは無しで、0.15 mol/l H3PO4溶液の１リットルを0.25 mol/l Ca(OH)2懸濁液１リットルへ室温で添加する。Ca(OH)2懸濁液へ分散するChSの濃度は2.5 mg/mlとし、10 wt%に調整する。懸濁液の最終pHは約8に調整する。得られた沈殿物をろ過し、乾燥し、次いで、乳棒と乳鉢で15分間粉砕する。かくして、HAp又はHAp/ChS複合体(composite)粉末が得られる。 An example of a method for producing a porous scaffold material will be described below. HAp and HAp / ChS complexes are synthesized by the precipitation method (T. Ikoma et al .: Key Eng. Mater. Vol. 288-289, (2005), pp. 159). Pure CaCO 3 powder is calcined at 1050 ° C. for 3 hours. The obtained CaO powder is hydrated with distilled water at 300 ° C. to produce Ca (OH) 2 . Add 1 liter of 0.15 mol / l H 3 PO 4 solution to 1 liter of 0.25 mol / l Ca (OH) 2 suspension at room temperature with or without the addition of ChS (Seikagaku, from bovine trachea) . The concentration of ChS dispersed in the Ca (OH) 2 suspension is 2.5 mg / ml and adjusted to 10 wt%. The final pH of the suspension is adjusted to about 8. The resulting precipitate is filtered, dried and then ground with a pestle and mortar for 15 minutes. Thus, HAp or HAp / ChS composite powder is obtained.
得られた粉末の結晶状態はＸ線回折(XRD)法及びフーリエ変換赤外分光学(FT-IR)で分析できる。粒子サイズの分布はイソプロピルアルコール中でレーザー回折法により測定できる。HAs/ChS中のChSの量は、熱重量測定分析(TG)により評価できる。粒子の形態学は走査型電子顕微鏡(SEM)で観察できる。 The crystalline state of the obtained powder can be analyzed by X-ray diffraction (XRD) method and Fourier transform infrared spectroscopy (FT-IR). The particle size distribution can be measured by laser diffraction in isopropyl alcohol. The amount of ChS in HAs / ChS can be evaluated by thermogravimetric analysis (TG). The morphology of the particles can be observed with a scanning electron microscope (SEM).
多孔質足場材料を以下のように加工する。1 wt%のアルカリ可溶性コラーゲン(豚の皮膚由来)をリン酸緩衝液(pH 7.2, 100 mM)に溶解し、その後、HAp/ChS又はHAp粉末をコラーゲン溶液に混合する。添加するHApの量は、各懸濁液について、1 wt%に固定する。1 gの懸濁液を24ウェルプレートに注ぎ、-20℃に凍結させ、その後、多孔質足場材料を得るために凍結乾燥する。多孔質足場材料のコラーゲン分子を真空中で24時間130℃にてdehydrothermal処理により架橋させる。多孔質足場材料はSEMで観察することができる。機械的性質は、4℃で一晩リン酸緩衝食塩水(PBS)に浸漬した後、PBS中で行う圧縮試験により、特性付けすることができる。足場材料は70%歪まで圧縮する。足場材料の圧縮係数は、得られた圧力-歪曲線から計算することができる。 The porous scaffold material is processed as follows. 1 wt% of alkali-soluble collagen (from pig skin) is dissolved in phosphate buffer (pH 7.2, 100 mM), and then HAp / ChS or HAp powder is mixed into the collagen solution. The amount of HAp added is fixed at 1 wt% for each suspension. 1 g of the suspension is poured into a 24-well plate and frozen at −20 ° C. and then lyophilized to obtain a porous scaffold material. The collagen molecules of the porous scaffold material are crosslinked by dehydrothermal treatment at 130 ° C. for 24 hours in a vacuum. The porous scaffold material can be observed by SEM. Mechanical properties can be characterized by a compression test performed in PBS after immersion in phosphate buffered saline (PBS) overnight at 4 ° C. The scaffold material compresses to 70% strain. The compression factor of the scaffold material can be calculated from the resulting pressure-strain curve.
コラーゲンとしては、動物等から抽出したものを使用することができ、由来する動物の種、組織部位等は特に限定されない。例えば、哺乳動物（例えば、ウシ、ブタ、ウマ、ウサギ、ネズミなど）や鳥類（例えば、ニワトリなど）の皮膚、骨、軟骨、腱、臓器などから得られるコラーゲンを使用できる。また、魚類（例えば、タラ、ヒラメ、カレイ、サケ、マス、マグロ、サバ、タイ、イワシ、サメなど）の皮、骨、軟骨、ひれ、うろこ、臓器などから得られるコラーゲン様タンパク質を使用してもよい。コラーゲンの抽出方法は特に限定されない。コラーゲンは天然のものでも、遺伝子組み換え技術により得られたものでもよい。また、アテロコラーゲンであってもよい。 As the collagen, those extracted from animals and the like can be used, and the species, tissue site, and the like of the derived animals are not particularly limited. For example, collagen obtained from the skin, bone, cartilage, tendon, organ, etc. of mammals (eg, cows, pigs, horses, rabbits, mice, etc.) and birds (eg, chickens, etc.) can be used. Also, using collagen-like proteins obtained from the skin, bones, cartilage, fins, scales, organs, etc. of fish (eg cod, flounder, flounder, salmon, trout, tuna, mackerel, Thailand, sardines, sharks, etc.) Also good. The method for extracting collagen is not particularly limited. Collagen may be natural or obtained by genetic recombination techniques. Moreover, atelocollagen may be sufficient.
ヒドロキシアパタイトとしては、種々の合成法で調製したものを使用することができ、結晶性や種々のイオンでの置換性は特に限定されない。例えば、カリウムイオン、ナトリウムイオン、マグネシウムイオン、ストロンチウムイオン、フッ化物イオン、炭酸イオンなどで結晶構造サイトが一部置換されたヒドロキシアパタイトを使用してもよい。 As the hydroxyapatite, those prepared by various synthetic methods can be used, and the crystallinity and the substitution with various ions are not particularly limited. For example, hydroxyapatite in which the crystal structure site is partially substituted with potassium ion, sodium ion, magnesium ion, strontium ion, fluoride ion, carbonate ion or the like may be used.
本発明の軟骨再生方法において使用するヒト軟骨膜細胞は、ヒト軟骨組織に由来する細胞であって、軟骨細胞に分化しうるものであればよい。ヒト軟骨膜組織に由来する細胞であって、軟骨細胞に分化しうるものは、軟骨幹及び／又は前駆細胞であると考えられる。軟骨膜組織は、耳介や肋軟骨などの軟骨組織を構成する組織の一部であって、軟骨膜を含む部分である。具体的には、最外層と線維芽細胞層（軟骨膜細胞層）を含む組織、最外層と線維芽細胞層（軟骨膜細胞層）と最内層を含む組織である。本発明で使用する細胞が由来するヒト軟骨膜組織は、最外層及び線維芽細胞層からなるとよく、最外層、線維芽細胞層及び最内層からなってもよい。軟骨膜組織は、ヒト、特に軟骨移植を必要とする患者から採取したものであるとよい。 The human perichondrial cell used in the cartilage regeneration method of the present invention may be any cell derived from human cartilage tissue and capable of differentiating into chondrocytes. Cells derived from human perichondrial tissue that can differentiate into chondrocytes are considered to be cartilage stem and / or progenitor cells. The perichondrial tissue is a part of a tissue constituting the cartilage tissue such as the auricle and the costal cartilage and includes the perichondrium. Specifically, a tissue including an outermost layer and a fibroblast layer (perichondrial cell layer), and a tissue including an outermost layer, a fibroblast layer (perichondrial cell layer) and an innermost layer. The human perichondrium tissue from which the cells used in the present invention are derived may be composed of an outermost layer and a fibroblast layer, and may be composed of an outermost layer, a fibroblast layer and an innermost layer. The perichondrial tissue may be collected from humans, particularly patients who require cartilage transplantation.
軟骨膜組織に由来する細胞は、軟骨膜組織から単離した細胞であっても、その細胞を継代培養して得られた細胞であってもよい。 The cell derived from the perichondrial tissue may be a cell isolated from the perichondrial tissue or a cell obtained by subculturing the cell.
軟骨膜組織に由来する細胞を得るには、軟骨膜組織を採取し、採取した組織から細胞を分離するとよい。軟骨膜組織の採取は、ピンセットとハサミなどの鋭的な器具を用いてもよいし、剥離子などの鈍的な器具を用いてもよい。軟骨膜組織から細胞を分離するには、軟骨膜組織を一定の条件下でコラゲナーゼ処理をし（例えば、0.1-0.3%コラゲナーゼ、37℃、1-3時間）、その後遠心分離する（例えば、1500 rpm/5 minで２回）とよい。これらの処理条件は適宜変更でき、その変更されたものも本発明の範囲内にある。軟骨膜組織から分離した細胞を培養することにより、細胞を増殖させ、さらには軟骨細胞に分化させることができる。 In order to obtain cells derived from the perichondrial tissue, it is preferable to collect the perichondrial tissue and separate the cells from the collected tissue. For collecting the perichondrial tissue, a sharp instrument such as tweezers and scissors may be used, or a blunt instrument such as an exfoliator may be used. To separate cells from the perichondrial tissue, the perichondrium tissue is treated with collagenase under certain conditions (eg, 0.1-0.3% collagenase, 37 ° C., 1-3 hours), and then centrifuged (eg, 1500). 2 times at rpm / 5 min). These processing conditions can be changed as appropriate, and those changes are also within the scope of the present invention. By culturing cells separated from the perichondrial tissue, the cells can be proliferated and further differentiated into chondrocytes.
軟骨膜組織から分離した細胞を初代培養及び継代培養するには、血清（例えば、10%のFetal Bovine Serum(FBS)、移植の対象となる患者由来の血清）を添加したDulbecco`s Modified Eagles`s Medium/Nutrient Mixture F12(DMEM/F12)もしくはNutrient Mixture
F-12 Ham(F-12 Ham)中にて、約37℃で培養するとよい。培地は2-4日毎に交換するとよい。
For primary and subculture of cells isolated from perichondrial tissue, Dulbecco`s Modified Eagles supplemented with serum (eg, 10% Fetal Bovine Serum (FBS), serum from the patient to be transplanted) `s Medium / Nutrient Mixture F12 (DMEM / F12) or Nutrient Mixture
Incubate in F-12 Ham (F-12 Ham) at about 37 ° C. The medium should be changed every 2-4 days.
軟骨細胞は、増殖させるために長期間の培養が困難であるが、軟骨膜細胞は軟骨細胞と比較して長期間の増殖培養が可能である。 Chondrocytes are difficult to culture for a long time in order to proliferate, but perichondrial cells can be proliferated and cultured for a long time compared to chondrocytes.
軟骨膜組織から分離した細胞を軟骨細胞に分化させるには、血清を含む培地、例えば、DEME/F12、血清（例えば、10%のFBS、移植の対象となる患者由来の血清）、抗生物質及び抗ミトティック剤を含有する培地中にて、約37℃で培養するとよい。抗生物質と抗ミトティック剤の両方を含む薬剤としては、antibiotic antimycotic solution SIGMA A5955などを使用することができる。培地には、40〜60 μg/mlのデキサメタゾン（Dexamethasone）及び／又は30〜60 μg/mlのL-アスコルビン酸（L-ascorbic acid）をさらに添加してもよい。また、インスリン様成長因子（例えば、5〜10 ng/mlのInsulinlike growth factor-1(IGF-1)、、5〜10 ng/mlの塩基性線維芽細胞成長因子（basic Fibroblast growth factor(bFGF)）などをさらに添加してもよい。その他に、5〜10 ng/mlのインスリン（Insulin）などを添加してもよい。培地交換は2-3日毎にするとよい。 To differentiate cells isolated from perichondrial tissue into chondrocytes, a medium containing serum, such as DEME / F12, serum (eg, 10% FBS, serum from the patient to be transplanted), antibiotics and The culture may be performed at about 37 ° C. in a medium containing an antimitotic agent. Antibiotic antimycotic solution SIGMA A5955 etc. can be used as a medicine containing both antibiotics and antimitotics. To the medium, 40 to 60 μg / ml dexamethasone (Dexamethasone) and / or 30 to 60 μg / ml L-ascorbic acid may be further added. Insulin-like growth factor (for example, 5-10 ng / ml insulin-like growth factor-1 (IGF-1), 5-10 ng / ml basic fibroblast growth factor (basic fibroblast growth factor (bFGF) In addition, 5 to 10 ng / ml insulin (Insulin), etc. may be added, and the medium should be changed every 2-3 days.
軟骨膜組織由来の細胞を遠心管培養することにより細胞塊を形成させることができる。例えば、5ng/mlのInsulinlike growth factor-1(IGF-1)、5ng/mlのbasic Fibroblast growth factor(bFGF)、40ng/mlのDexamethasone、L-ascorbic acid、1%Antibiotic antimicotic solution、Insulin・Transferrin・Serine(ITS)を含むDMEM/F12の無血清培地中にて、約37℃で遠心管培養で2-4週間培養すると、細胞塊が形成する。 A cell mass can be formed by culturing cells derived from perichondrial tissue in a centrifuge tube. For example, 5 ng / ml Insulinlike growth factor-1 (IGF-1), 5 ng / ml basic Fibroblast growth factor (bFGF), 40 ng / ml Dexamethasone, L-ascorbic acid, 1% Antibiotic antimicotic solution, Insulin Transferrin When cultured in a serum-free medium of DMEM / F12 containing Serine (ITS) at about 37 ° C. for 2-4 weeks in a centrifuge tube, a cell mass is formed.
また、軟骨膜組織由来の細胞を平板培養で単層化又は重層化することができる。例えば、DMEM/F12に10% FBSと1%Antibiotic antimicotic solutionを含んだ培地もしくはDMEM/F12に10% FBS、1%Antibiotic antimicotic solution、5ng/mlのIGF-1、5ng/mlのbFGF、40ng/mlのDexamethasoneを含んだ培地を用いて平板培養し、１週間毎に重層化すると、基質（例えば、プロテオグリカン）産生能が高まる。重層化の回数は、用途や必要とされる組織の大きさなどによって異なるが、通常、3〜5回が適当である。 In addition, cells derived from perichondrial tissue can be monolayered or stratified by plate culture. For example, a medium containing 10% FBS and 1% Antibiotic antimicotic solution in DMEM / F12 or 10% FBS, 1% Antibiotic antimicotic solution in DMEM / F12, 5 ng / ml IGF-1, 5 ng / ml bFGF, 40 ng / When the plate is cultured using a medium containing ml of Dexamethasone and layered every week, the substrate (for example, proteoglycan) production ability increases. The number of stratifications varies depending on the application and the size of the required tissue, but usually 3 to 5 times is appropriate.
上記培地の組成及び成分含量は適宜変更でき、その変更されたものも本発明の範囲内にある。 The composition and component content of the medium can be changed as appropriate, and those changes are also within the scope of the present invention.
軟骨膜組織由来の細胞は、CD44+CD90+の表現型を有するものであることが好ましい。 Cells derived from the perichondrial tissue preferably have a CD44 + CD90 + phenotype.
軟骨膜組織由来の細胞の培養は、三次元擬微小重力培養であることが好ましい。軟骨膜組織由来の細胞を擬微小重力環境下（浮遊状態）で培養することにより、細胞に与えられる機械的刺激や損傷が強く、大きな組織を得ることができる。例えば、ＮＡＳＡが開発したガス交換機能を備えた回転式ＲＷＶ（Ｒｏｔａｔｉｎｇ−Ｗａｌｌ Ｖｅｓｓｅｌ）バイオリアクターによる培養法が利用可能である。 The culture of perichondrial tissue-derived cells is preferably a three-dimensional pseudo-microgravity culture. By culturing cells derived from perichondrium tissue in a pseudo-microgravity environment (floating state), a large tissue can be obtained due to strong mechanical stimulation and damage given to the cells. For example, a culture method using a rotating RWV (Rotating-Wall Vessel) bioreactor having a gas exchange function developed by NASA can be used.
本発明の軟骨再生方法は、ヒト以外の生物由来の軟骨膜細胞を用いることにより、ヒト以外の生物の軟骨再生にも応用可能である。 The method for regenerating cartilage of the present invention can be applied to regeneration of cartilage of organisms other than humans by using perichondrial cells derived from organisms other than humans.
また、本発明は、ヒドロキシアパタイトとコラーゲンを含む多孔質足場材料及びヒト軟骨膜細胞を含む、軟骨再生のための組成物を提供する。 The present invention also provides a composition for cartilage regeneration comprising a porous scaffold material containing hydroxyapatite and collagen and human perichondrial cells.
ヒドロキシアパタイトとコラーゲンを含む多孔質足場材料及びヒト軟骨膜細胞は上記の通りである。 The porous scaffold material containing hydroxyapatite and collagen and human perichondrial cells are as described above.
本発明の組成物は、さらに、ヒト軟骨膜細胞が産生する基質（例えば、aggrecan(ACAN) 、versican(CSPG2)、elastin (ELN)、alpha 1 type II collagen (COL2A1)、fibrillin 1 (FBN1)など）などを含んでもよい。 The composition of the present invention further comprises a substrate produced by human perichondrial cells (eg, aggrecan (ACAN), versican (CSPG2), elastin (ELN), alpha 1 type II collagen (COL2A1), fibrillin 1 (FBN1), etc. ) Or the like.
本発明の軟骨再生法及び組成物は以下の応用が可能である。 The cartilage regeneration method and composition of the present invention can be applied as follows.
・先天奇形
鞍鼻/短鼻、Binder症候群、***口蓋裂に対する隆鼻術あるいはTreacher-Collins症候群、nager症候群に対する眼窩周囲の陥凹修正術は技術的に臨床応用可能である。
・ Congenital malformations: Rhinoplasty for vomeronasal / short nose, Binder syndrome, cleft lip and / or palate or Treacher-Collins syndrome, correction of periorbital depression for nager syndrome is technically applicable clinically.
小耳症に対する耳介再建術は開発段階である。 Auricular reconstruction for microtia is in development.
・美容整形
審美的改善のための治療、例えば隆鼻術（鼻を高くする）、オトガイ形成術（アゴを出す）、顔面の小陥凹形成術、顔面左右非対称の修正術、眼瞼周囲の修正術は技術的に臨床応用可能である。
・外傷
顔面の小陥凹形成術、顔面左右非対称の修正術などは技術的に臨床応用可能である。
・ Cosmetics Treatment for aesthetic improvement, such as bulging rhinoplasty (raising the nose), genioplasty (making the chin), facial concavity shaping, facial asymmetrical correction, and correction around the eyelids Technically applicable clinically.
-Trauma Facial minor concavities and facial asymmetry correction techniques are clinically applicable.
以下、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.
〔実施例１〕耳介軟骨膜由来の新規軟骨幹/前駆細胞を用いたヒト弾性軟骨再構築法
Abstract
頭蓋・顎・顔面領域の組織変形に対する現在の標準的な治療法では、広範な変形に対する治療は困難であり、長期的な形態保持性の観点からも満足の行く臨床成績が得られていない。そこで、これらの課題を克服しうる新たな治療法の一つとして、組織再生工学を用いたヒト弾性軟骨の臨床的再構築法の開発が待望されている。我々は、低侵襲操作で採取可能なヒト耳介軟骨膜中から高い増殖能、多分化能、自己複製能などの特徴を有する軟骨幹/前駆細胞を分離することに成功した。このヒト軟骨幹/前駆細胞は、in
vitroで耳介軟骨細胞と同等の軟骨基質産生を行うことが確認されただけでなく、重症免疫不全マウスへの皮下移植により長期形態保持性に優れたヒト弾性軟骨組織を再構築した。さらに、足場材料の新規開発を行い、本軟骨幹/前駆細胞と組み合わせることで、軟骨基質産生を行う成熟軟骨細胞が均一(Homogenous)に存在する大型のヒト弾性軟骨組織を再構築させることに成功した。我々が開発したヒト弾性軟骨再構築法は、次世代の弾性軟骨再生治療の実現にとって革新的な方法となる可能性がある。
[Example 1] Reconstruction of human elastic cartilage using novel cartilage stem / progenitor cells derived from auricular perichondrium
Abstract
The current standard treatments for tissue deformation in the skull, jaw, and face region are difficult to treat for widespread deformation, and satisfactory clinical results are not obtained from the viewpoint of long-term shape retention. Therefore, development of a clinical reconstruction method for human elastic cartilage using tissue regeneration engineering is awaited as one of the new treatment methods that can overcome these problems. We succeeded in isolating cartilage stem / progenitor cells with high proliferative ability, pluripotency, and self-renewal ability from human auricular perichondrium that can be collected by minimally invasive procedures. This human cartilage stem / progenitor cell
In addition to being confirmed to produce cartilage matrix equivalent to auricular chondrocytes in vitro, human elastic cartilage tissue excellent in long-term morphology retention was reconstructed by subcutaneous implantation into severely immunodeficient mice. Furthermore, by developing new scaffold materials and combining them with the cartilage stem / progenitor cells, we succeeded in reconstructing a large human elastic cartilage tissue in which mature chondrocytes that produce cartilage matrix are uniformly present (homogenous). did. The human elastic cartilage reconstruction method that we have developed may be an innovative method for realizing the next generation of elastic cartilage regeneration therapy.
Introduction
頭蓋・顎・顔面領域の先天奇形や外傷に起因する組織変形に対する新しい治療法の開発は、全世界で１００万人以上の患者に待ち望まれている極めて重要な臨床的解決課題である1。現在の標準的な治療法は、自家軟骨/骨組織を移植する方法や合成高分子化合物などの医用材料を移植する方法である2-5。しかし、自己組織移植では、採取量の制限と採取部位の侵襲が軟骨/骨組織移植に共通した問題である。また、軟骨組織移植に伴う経年的な組織変形と吸収や、骨組織移植に伴う経月的な組織吸収も極めて大きな問題となっており臨床的に満足のいく長期成績が得られていない6-10。医用材料を移植する方法においても、それらが人体にとり異物であることから、感染や炎症、それらに起因する皮膚穿孔などが生じることが知られており、これらの問題が未解決である。このような問題点を克服することの可能な新しい治療法として、組織再生工学を用いたヒト弾性軟骨の臨床的再構築法の開発が切望されている。
Introduction
The development of new treatments for tissue deformities caused by congenital malformations and trauma in the skull, jaw, and face is an extremely important clinical solution that is awaited by more than 1 million patients worldwide 1 . Current standard therapies are transplanting autologous cartilage / bone tissue or medical materials such as synthetic polymer compounds 2-5 . However, in autologous tissue transplantation, the limitation of the collection amount and the invasion of the collection site are common problems in cartilage / bone tissue transplantation. In addition, and absorption and secular organization deformation associated with cartilage tissue transplantation, not long-term results can be obtained to go clinically satisfactory after month organization absorption associated with bone tissue transplantation has become a very big problem 6 10 . Also in the method of transplanting medical materials, since these are foreign substances in the human body, it is known that infection, inflammation, and skin perforation resulting from them occur, and these problems are unsolved. As a new treatment method capable of overcoming such problems, development of a clinical reconstruction method of human elastic cartilage using tissue regeneration engineering is eagerly desired.
ヒト弾性軟骨の再構築法に適応可能な細胞源として、幾つかの可能性が示唆されている11-13。ヒト耳介軟骨細胞は良好な基質産生能などの優位性を有するものの、採取部位への侵襲に加え、自己複製能を有する幹細胞が存在しないために細胞寿命に起因する長期的な組織維持の困難性などの問題点を抱えている。骨髄由来のヒト間葉系幹細胞は、これらの諸問題を解決できる可能性を持つ細胞の一つであるが、骨髄穿刺の侵襲が大きいこと、成熟軟骨細胞への分化能が極めて低いこと、血管侵入や石灰沈着をきたすことなどの様々な問題を抱えているため実用化の可能性は低い14-16。他にも脂肪組織由来のヒト間葉系幹細胞など候補となる細胞は存在するものの、いずれも成熟軟骨細胞への分化能力が低く、弾性軟骨における細胞外マトリックスの産生能は全く確認されていないことから、ヒト弾性軟骨の再構築法に応用可能な優れた細胞源は見いだされていないのが現状である17-19。 Several possibilities have been suggested as a source of cells that can be adapted for the reconstruction of human elastic cartilage 11-13 . Although human auricular chondrocytes have advantages such as good matrix production ability, in addition to invasion at the collection site, there is no self-replicating stem cell, so long-term tissue maintenance due to cell life is difficult I have problems such as sex. Bone marrow-derived human mesenchymal stem cells are one of the cells that have the potential to solve these problems. However, the bone marrow puncture is highly invasive, the ability to differentiate into mature chondrocytes is extremely low, and blood vessels possibility of practical use because of various problems, such as that leading to penetration and calcification is low 14-16. Although there are other candidate cells such as human mesenchymal stem cells derived from adipose tissue, all have low ability to differentiate into mature chondrocytes, and no ability to produce extracellular matrix in elastic cartilage has been confirmed from excellent cell source that can be applied to reconstruction method of human elastic cartilage it is has not yet been found 17-19.
組織再生工学的手法を用いた頭蓋・顎・顔面領域における弾性軟骨の再生治療においては、審美的観点から再構築された軟骨組織の形態を再現するための足場材料（スキャフォールド）の開発が重要である20。足場材料については、これまでにPLA(poly lactic acid)、PGA(poly glycolic acid)などの合成高分子や、コラーゲンスポンジなどの生体由来の高分子材料が開発されているものの、臨床応用は成功していない21-23。高品質の医用足場材料は、一般的に細胞や栄養因子の浸透が可能な連通性(porosity)、高圧縮弾性率(elasticity)、優れた生分解性などの特徴を兼ね備えている必要があるが、これらの諸条件を十分に満たす足場材料は未だに開発されていない。さらに、優れた弾性軟骨組織を再構築するためには、これらの諸条件に加え、幹/前駆細胞の増殖・分化に適した細胞外環境を提供し、Homogenousに成熟軟骨細胞が局在することのできる生分解性足場材料の開発が不可欠である。 In regenerative treatment of elastic cartilage in the skull, jaw, and face using tissue regeneration engineering techniques, it is important to develop scaffolds (scaffolds) to reproduce the reconstructed cartilage tissue morphology from an aesthetic point of view 20 is . As for scaffold materials, synthetic polymers such as PLA (poly lactic acid) and PGA (poly glycolic acid) and biological polymer materials such as collagen sponge have been developed, but clinical application has been successful. Not 21-23 . High-quality medical scaffold materials generally need to have characteristics such as porosity, high compressibility (elasticity), and excellent biodegradability that allow penetration of cells and nutrient factors Scaffold materials that sufficiently satisfy these conditions have not yet been developed. Furthermore, in order to reconstruct an excellent elastic cartilage tissue, in addition to these conditions, it provides an extracellular environment suitable for stem / progenitor cell proliferation and differentiation, and mature chondrocytes are localized in homogenous. It is essential to develop biodegradable scaffold materials that can be used.
ヒト弾性軟骨の再生治療を実現するためには、低侵襲操作による採取が可能で、高い増殖活性、成熟軟骨細胞への効率の良い分化能、自己複製能を持つ適切なヒト幹/前駆細胞を同定し、これらの分離法・培養法・分化誘導法などに関する様々な細胞操作技術を開発することが必要である。さらに、耳介軟骨等の複雑な形状をもつ弾性軟骨組織の再構築を実現化するためには、ヒト幹/前駆細胞を最適化された医用足場材料とともに移植するための手法が開発されることが必須である。 In order to achieve regenerative treatment of human elastic cartilage, appropriate human stem / progenitor cells that can be collected by minimally invasive procedures, have high proliferative activity, efficient differentiation into mature chondrocytes, and self-renewal ability It is necessary to identify and develop various cell manipulation techniques related to these separation methods, culture methods, differentiation induction methods and the like. Furthermore, in order to realize the reconstruction of elastic cartilage tissue with a complicated shape such as auricular cartilage, a method for transplanting human stem / progenitor cells with optimized medical scaffold materials should be developed. Is essential.
本研究では、全く解明の進んでいないヒト弾性軟骨における幹/前駆細胞の分離・同定を、低侵襲操作で臨床的に採取が可能な耳介軟骨膜部を対象として試みた。また、分離したヒト軟骨幹/前駆細胞の操作技術を検討し、臨床応用の可能な優れた弾性軟骨再構築法の開発を行った。さらに、我々が新規に開発した革新的な生分解性足場材料がヒト弾性軟骨の再構築に有用か否かを検討した。 In this study, we attempted to isolate and identify stem / progenitor cells in human elastic cartilage, which has not been elucidated at all. In addition, we investigated the manipulation technique of the isolated human cartilage stem / progenitor cells and developed an excellent elastic cartilage reconstruction method that can be applied clinically. Furthermore, we investigated whether the newly developed innovative biodegradable scaffold material is useful for the reconstruction of human elastic cartilage.
RESULTS
High proliferative capacity of human perichondrocytes
我々は高い増殖能、多分化能、自己複製能、組織再構築能などの幹細胞としての特徴を有する細胞集団は耳介軟骨膜部に存在するものと仮定した。これらの仮説を実証するため、まず、マウスの耳介軟骨幹/前駆細胞を同定するために、幹細胞を同定するための一つの手法である5-bromo-2`-deoxyuridine (BrdU)によるLabel-retaining cells(LRC)の観察を行った。BrdUは細胞周期の合成期にチミジンの代わりに取り込まれ、2〜3回の***までは検出可能であるが、それ以上の***を起こした細胞では検出が困難になる。一般的に、幹細胞は通常、***休止状態にあり、傷害時における組織修復など必要に応じて細胞***を起こすため、その***頻度は極めて低いと考えられている。そのため、幹細胞の***時にBrdUが取り込まれた場合、幹細胞や自己複製で生じた娘細胞では長期間にわたりBrdUが残存し続けることになる。この様なBrdUにより長期間ラベルされた細胞がLRCとして定義されている。幹細胞は通常の状態ではほとんど***しないと考えられるため、我々は胎生期にBrdUを取り込ませ1年間にわたり観察した。5-bromo-2`-deoxyuridine
（BrdU）（50μg/g body WT )を、エーテルで麻酔した妊娠17.5日目の母体マウスに12時間毎、計6回腹腔内投与した。妊娠マウスでは、生まれた仔を各週齢（新生仔〜2週齢、4週齢、24週齢、48週齢）の段階において解析に用いた。軟骨膜のBrdU陽性細胞は、BrdU投与直後の新生仔から2週齢まで減少したが、1年後までわずかながら存在した(図7b)。軟骨膜と軟骨における一視野の背景細胞数で割ったBrdU陽性細胞数をLabel-retaining cells（LRC）残存比率とした。そして、100倍下の5視野の平均値から算出し、BrdU
labeling Index (LI)とした。生後0,3日,1,2,4,24.48週間目のLIは86.6±2.8%、34±7.2%、3.8±1.3%、2.1±1.5%、0.3±0.2%、0.1±0.05%、0.08±0.06%であった。一方、軟骨のLRCは減少し続け、4週間目までに認められなくなった。軟骨の生後0,3日,1,2,4,24.48週間目のLIは86.4±3.3%、29±3.8%、0.93±0.4%、0.67±0.4%、0.0±0.0%、0.0±0.0%、0.0±0.0%であった(図7a)。
RESULTS
High proliferative capacity of human perichondrocytes
We hypothesized that a cell population with high proliferative ability, pluripotency, self-renewal ability, tissue remodeling ability, and other stem cell characteristics exists in the auricular perichondrium membrane region. In order to demonstrate these hypotheses, first, in order to identify mouse auricular cartilage stem / progenitor cells, Label- by 5-bromo-2`-deoxyuridine (BrdU), a technique for identifying stem cells, was used. Retaining cells (LRC) were observed. BrdU is taken up in place of thymidine during the synthesis of the cell cycle and can be detected up to 2 to 3 divisions, but is difficult to detect in cells that have undergone further division. In general, stem cells are normally in a divisional quiescent state and cause cell division as needed, such as tissue repair at the time of injury, so the frequency of division is considered to be extremely low. Therefore, when BrdU is taken up during the division of stem cells, BrdU continues to remain for a long time in the stem cells and daughter cells generated by self-replication. Cells labeled for a long time with such BrdU are defined as LRC. Since stem cells are thought to divide little under normal conditions, we took BrdU uptake during the embryonic period and observed it for one year. 5-bromo-2`-deoxyuridine
(BrdU) (50 μg / g body WT) was intraperitoneally administered every 12 hours to a mother mouse on day 17.5 of pregnancy anesthetized with ether. In pregnant mice, born pups were used for analysis at the age of each week (newborn to 2 weeks old, 4 weeks old, 24 weeks old, 48 weeks old). BrdU positive cells in the perichondrium decreased from neonates immediately after BrdU administration to 2 weeks of age, but remained slightly until one year later (FIG. 7b). The number of BrdU positive cells divided by the number of background cells in one field of view in the perichondrium and cartilage was defined as the residual ratio of label-retaining cells (LRC). And calculated from the average value of 5 fields of view 100 times lower, BrdU
Labeling Index (LI). LI at 0,3 days, 1,2,4,24.48 weeks after birth is 86.6 ± 2.8%, 34 ± 7.2%, 3.8 ± 1.3%, 2.1 ± 1.5%, 0.3 ± 0.2%, 0.1 ± 0.05%, 0.08 ± It was 0.06%. On the other hand, the LRC of cartilage continued to decrease and disappeared by 4 weeks. LI of cartilage 0,3 days, 1,2,4,24.48 weeks is 86.4 ± 3.3%, 29 ± 3.8%, 0.93 ± 0.4%, 0.67 ± 0.4%, 0.0 ± 0.0%, 0.0 ± 0.0%, 0.0 ± 0.0% (FIG. 7a).
次に、ヒト小耳症患者から摘出した残存耳介軟骨を対象として、ヒト弾性軟骨組織における幹/前駆細胞の分離・同定を試みた。残存耳介軟骨を提供した患者の性別に偏りはなく、平均年齢は10.6±1.4歳であった（Supplementary Table 1）。残存耳介軟骨を外科的に摘出後、軟骨膜部、軟骨-軟骨膜移行部、軟骨実質部の三層に分離し、各々から単離した細胞をそれぞれ培養した（図1abc）。まず各層由来細胞の増殖能を比較するため、低密度培養（52 cells/cm2）によるクローン性コロニー形成能の解析を行った。培養4週後、各細胞からクローン性コロニーが形成された（図1d）。形成されたクローン性コロニー数は、播種細胞500個あたり軟骨膜細胞で23.9±4.5個、軟骨膜-軟骨移行部細胞で9.9±6.8個、軟骨細胞で2.3±0.4個であり、軟骨膜細胞は他と比べて極めて高いコロニー形成能を有していた（図1e）。 Next, separation / identification of stem / progenitor cells in human elastic cartilage tissue was attempted using residual auricular cartilage removed from a patient with microtia. There was no gender bias in the patients who provided residual auricular cartilage, with an average age of 10.6 ± 1.4 years (Supplementary Table 1). The remaining auricular cartilage was surgically removed and then separated into three layers of perichondrium, cartilage-perichondrium transition, and cartilage parenchyma, and the cells isolated from each were cultured (FIG. 1abc). First, in order to compare the proliferative ability of cells derived from each layer, the ability to form clonal colonies by low density culture (52 cells / cm 2 ) was analyzed. After 4 weeks of culture, clonal colonies were formed from each cell (FIG. 1d). The number of clonal colonies formed was 23.9 ± 4.5 perichondrial cells per 500 seeded cells, 9.9 ± 6.8 perichondrial-cartilage transition cells, 2.3 ± 0.4 chondrocytes, Compared with others, it had a very high ability to form colonies (FIG. 1e).
軟骨膜細胞の長期的な増殖能を継代培養系で解析した。形態学的には、各層由来細胞は長期培養後（12w〜）には扁平化し、線維芽細胞様の形態を有するようになった（図1f）。196日間にわたって14回の継代培養を行った結果、9.42×103個の軟骨膜細胞は、1.20×1027個まで約1.27×1022倍に増殖した。一方、9.42×103個の軟骨細胞は、1.26×1024個まで約1.30×1019倍に増殖した。すなわち、軟骨膜細胞は軟骨細胞に比べ約949倍の子孫細胞を生み出す能力を有していることが判明し、有意に高い増殖活性をもつことが明らかとなった（図1g）。 The long-term proliferative ability of perichondrial cells was analyzed in a subculture system. Morphologically, the cells derived from each layer flattened after long-term culture (from 12w) and had a fibroblast-like morphology (FIG. 1f). As a result of performing subculture for 14 times over 196 days, 9.42 × 10 3 perichondrocytes proliferated approximately 1.27 × 10 22 times up to 1.20 × 10 27 cells. On the other hand, 9.42 × 10 3 chondrocytes grew about 1.30 × 10 19 times up to 1.26 × 10 24 cells. That is, it was found that perichondrocytes have the ability to generate progeny cells about 949 times that of chondrocytes, and it was revealed that it has significantly higher proliferative activity (FIG. 1g).
Flow-cytometric enrichment of putative human cartilage stem/progenitor cells by
CD44/CD90
耳介軟骨幹/前駆細胞を効率良く単離するため、まず、マウスにおいてにフローサイトメトリーを用いた耳介軟骨幹細胞のprospective isolationを目指して、既存の関節軟骨の表面抗原を参考に軟骨幹細胞特異的抗原のスクリーニングを行った。LRCが局在する部位に発現する表面抗原を耳介軟骨幹/前駆細胞特異的抗原の候補とし、LRCとそれら特異的抗原の関連を組織学的に検討した結果、表面抗原の免疫染色においてCD44が軟骨膜特異的に発現していた(図8a)。しかも、BrdU投与後48週後に分離されたLRCはCD44を発現していた(図8b)。
Flow-cytometric enrichment of putative human cartilage stem / progenitor cells by
CD44 / CD90
In order to efficiently isolate the auricular cartilage stem / progenitor cells, we first aimed at the prospective isolation of the auricular cartilage stem cells using flow cytometry in mice, with reference to the surface antigens of existing articular cartilage, and specific for cartilage stem cells Antigenic antigens were screened. As a result of histological examination of the relationship between LRC and these specific antigens, the surface antigen expressed at the site where LRC is localized was selected as a candidate for auricular cartilage stem / progenitor cell specific antigen. Was expressed specifically in the perichondrium (FIG. 8a). Moreover, LRC isolated 48 weeks after BrdU administration expressed CD44 (FIG. 8b).
次に、ヒト軟骨幹/前駆細胞の特異的マーカーを同定することを目的として、フローサイトメトリーを用いた細胞表面抗原の発現解析を行った。造血幹細胞に発現していることが知られているCD34、c-kit(CD117)、間葉系幹細胞に発現していることが報告されているCD44、CD73、CD90、CD105、CD133、CD140a、CD146、CD271の発現を解析した(図2a) 24-28。これらの表面抗原のうち、CD44およびCD90の陽性率は軟骨細胞と軟骨膜細胞において大きな相違が観察された。すなわち、CD44陽性率は、軟骨細胞で35.4±7.3%であったのに対し、軟骨膜細胞では58.8±8.6%であった。CD90陽性率は、軟骨細胞で46.5±3.9%であったのに対し、軟骨膜細胞では63.0±1.7%であった（図2a）。 Next, for the purpose of identifying specific markers of human cartilage stem / progenitor cells, cell surface antigen expression analysis using flow cytometry was performed. CD34, c-kit (CD117) known to be expressed on hematopoietic stem cells, CD44, CD73, CD90, CD105, CD133, CD140a, CD146 reported to be expressed on mesenchymal stem cells CD271 expression was analyzed (FIG. 2a) 24-28 . Among these surface antigens, a large difference was observed in the positive rate of CD44 and CD90 in chondrocytes and perichondrial cells. That is, the CD44 positive rate was 35.4 ± 7.3% in chondrocytes, whereas it was 58.8 ± 8.6% in perichondrial cells. The CD90 positive rate was 46.5 ± 3.9% in chondrocytes, compared with 63.0 ± 1.7% in perichondrial cells (FIG. 2a).
CD44およびCD90の発現を指標として、ヒト軟骨膜細胞をCD44-CD90-、CD44-CD90+、CD44+CD90-、CD44+CD90+細胞に分画化した(図2b)。セルソーティングにより選択的に分離された各細胞分画のうち高いコロニー形成を行う細胞がいずれの分画中に濃縮されるかを検証するため、低密度培養系（52cells/cm2）でコロニーアッセイを行った。CD44+CD90+細胞は、他の細胞分画と比較してより高いクローン性コロニー形成能を有していた(図2c,d)。さらに、直径5mm以上の巨大なクローン性コロニー形成能を有する細胞の存在比率を算出すると、CD44-CD90-、CD44-CD90+、CD44+CD90-、CD44+CD90+細胞の順に有意に上昇し、極めて高い増殖能を有する細胞はCD44+CD90+分画中に最も高頻度に存在していることが判明した(図2 e)。以上から、CD44、CD90のいずれも幹/前駆細胞の純化に重要なマーカーとなることが推測された。 Using the expression of CD44 and CD90 as an index, human perichondrial cells were fractionated into CD44-CD90-, CD44-CD90 +, CD44 + CD90-, CD44 + CD90 + cells (FIG. 2b). Colony assay in low-density culture system (52cells / cm 2 ) to verify in which fraction the cells with high colony formation are concentrated in each cell fraction selectively separated by cell sorting Went. CD44 + CD90 + cells had a higher clonal colony forming ability compared to other cell fractions (FIGS. 2c, d). Furthermore, when calculating the abundance ratio of cells having a large clonal colony-forming ability with a diameter of 5 mm or more, CD44-CD90-, CD44-CD90 +, CD44 + CD90-, CD44 + CD90 + cells are significantly increased in this order and extremely high It was found that cells having proliferative ability were most frequently present in the CD44 + CD90 + fraction (FIG. 2e). From the above, it was speculated that both CD44 and CD90 are important markers for the purification of stem / progenitor cells.
In vitro elastic cartilage differentiation potential of human perichondrocytes
軟骨膜細胞の弾性軟骨への分化能を評価することを目的として、積層化培養法を用いて軟骨細胞への分化誘導を行った（図3a）。軟骨膜細胞は積層化を行うことによって、プロテオグリカン、Type
II collagen(Col 2)を産生する軟骨細胞へ分化することが確認された（図3b）。軟骨膜細胞から分化した軟骨細胞は種々のムコ多糖類を分泌するようになり、培養液は高い粘性を有する基質様性状へと変化した(Supplementary movie 1,2)。
In vitro elastic cartilage differentiation potential of human perichondrocytes
In order to evaluate the differentiation ability of perichondrial cells into elastic cartilage, differentiation into chondrocytes was induced using a layered culture method (Fig. 3a). Proteoglycan, Type by perforating perichondrocytes
Differentiation into chondrocytes producing II collagen (Col 2) was confirmed (FIG. 3b). Chondrocytes differentiated from perichondrial cells began to secrete various mucopolysaccharides, and the culture medium changed to a substrate-like property with high viscosity (Supplementary movies 1, 2).
軟骨細胞への分化能を定量的に検討するため、リアルタイムPCRを用いた弾性軟骨分化関連遺伝子の発現変化を解析した。軟骨膜細胞を積層化培養することにより、弾性軟骨に特徴的な基質であるversican(CSPG2)、elastin(ELN)、alpha 1 type II collagen (COL2A1)、fibrillin 1 (FBN1)遺伝子の発現レベルは、各々4.2倍、9.6倍、2.1倍、17.2倍に著名に上昇することが確認された（図3c）。一方、軟骨膜部に特徴的なalpha 1 type I collagen(COL1A1)の発現は0.18倍に低下した（図3c）。ELISAを用いて、弾性軟骨組織の細胞外マトリックスであるプロテオグリカン、エラスチン、コラーゲン産生能の解析を行ったところ、積層化した軟骨膜細胞においては各々17.5±4.3、235.6±19.9、61.8±7.5μg/mlの基質産生が確認された。驚くべき事に、この基質産生能は、同様に積層化した軟骨細胞のプロテオグリカン、エラスチン、コラーゲン産生能（各々19.0±1.3、234.0±16.3、55.8±4.9μg/ml）と同等であることが判明した（図3d）。 In order to quantitatively examine the differentiation potential into chondrocytes, we analyzed changes in the expression of elastic cartilage differentiation-related genes using real-time PCR. By layering culture of perichondrial cells, the expression levels of versican (CSPG2), elastin (ELN), alpha 1 type II collagen (COL2A1) and fibrillin 1 (FBN1) genes, which are characteristic substrates of elastic cartilage, It was confirmed that it increased prominently by 4.2 times, 9.6 times, 2.1 times, and 17.2 times, respectively (Fig. 3c). On the other hand, the expression of alpha 1 type I collagen (COL1A1), which is characteristic of the perichondrium, decreased 0.18 times (FIG. 3c). Using ELISA, the proteoglycan, elastin, and collagen production ability of the extracellular matrix of elastic cartilage tissue was analyzed. In the layered perichondrocytes, 17.5 ± 4.3, 235.6 ± 19.9, 61.8 ± 7.5 μg / ml substrate production was confirmed. Surprisingly, this matrix-producing ability was found to be equivalent to the proteoglycan, elastin, and collagen-producing ability (19.0 ± 1.3, 234.0 ± 16.3, and 55.8 ± 4.9 μg / ml, respectively) of similarly laminated chondrocytes (Figure 3d).
In vitro multi-differentiation and self-renewal capabilities of human
perichondrocytes
軟骨細胞への分化能に加え、軟骨膜細胞の脂肪および骨分化能の有無を検討した。脂肪分化誘導培地による3週間の培養により、軟骨膜細胞は卵円形の形態に変化し、Oil Red Oにて染色される脂肪滴を形成することが確認された。また、骨分化誘導培地による3週間の培養により、軟骨膜細胞はAlizarin Red Sにて染色されるCaを多量に産生することが確認された（図3e）。軟骨細胞を対象として同様の分化誘導を行ったところ、脂肪滴形成もCa沈着も認めなかったことから、軟骨膜細胞のみが軟骨分化能に加え脂肪・骨への多分化能をもつ事が示された（図3c）。
In vitro multi-differentiation and self-renewal capabilities of human
perichondrocytes
In addition to the ability to differentiate into chondrocytes, the presence or absence of fat and bone differentiation ability of perichondrial cells was examined. It was confirmed that the perichondrial cells changed to an oval shape and formed lipid droplets stained with Oil Red O after 3 weeks of culturing in the adipose differentiation induction medium. In addition, it was confirmed that perichondrial cells produced a large amount of Ca stained with Alizarin Red S by culturing for 3 weeks in a bone differentiation-inducing medium (FIG. 3e). When similar differentiation induction was performed on chondrocytes, neither lipid droplet formation nor Ca deposition was observed, indicating that only perichondrocytes have multipotency into fat and bone in addition to cartilage differentiation. (Figure 3c).
長期間にわたって、多分化能を有した幹/前駆細胞が自己複製により維持されていることを証明するため、継代培養を行った軟骨膜細胞を対象として多分化能の解析を行った。第3継代、7継代、10継代培養後の各軟骨膜細胞は、細胞化学染色、RT-PCRの結果より何れも軟骨、脂肪、骨への多分化能を維持していたことから、これらの細胞のうち少なくとも一部は自己複製能を有している事が示唆された(図9)。 In order to prove that stem / progenitor cells having pluripotency were maintained by self-replication over a long period of time, pluripotency analysis was performed on perichondrial cells that had been subcultured. From the results of cytochemical staining and RT-PCR, all the perichondrial cells after the 3rd, 7th and 10th passages maintained pluripotency into cartilage, fat and bone. Therefore, it was suggested that at least some of these cells have self-replicating ability (FIG. 9).
In vivo tissue reconstruction capability of human perichondrocytes
我々は高い軟骨細胞への分化能や自己複製能などの特徴を有している軟骨膜細胞を用いれば、in vivoにおいて優れた弾性軟骨再構築法を新規開発できるものと考えた。このアプローチの実現可能性を検討するため、ヒト軟骨膜細胞を軟骨分化誘導の後、粘性を帯びた培養上清とともに重症免疫不全マウスの皮下に移植した。尚、比較のためヒト軟骨細胞についても同様の手順で移植を行った。軟骨膜細胞は、移植3ヶ月目に軟骨様組織形成した(図4i)。組織学的解析から、軟骨膜細胞は、軟骨細胞と同様にin vivoで成熟軟骨細胞へと分化し、その産生基質であるプロテオグリカンや弾性線維に富む弾性軟骨組織を再構築することが判明した(図4i-p)。一方、免疫組織化学染色からは軟骨膜細胞より再構築された組織においてのみ、Col 2陽性弾性軟骨組織の周囲をType I collagen (Col 1)陽性の膜様組織が被覆していることが確認された(図4p)。長期間に渡って再構築組織が維持されていることを示すため、軟骨膜細胞を移植後6ヶ月並びに10ヶ月目に摘出した組織においても同様の解析を行った。いずれの時点においても摘出組織はCol 1陽性膜様組織で被覆されており、成熟軟骨細胞とその産生基質から構成される弾性軟骨組織であることが判明した(図10a-l)。一方、腫瘍形成や、骨髄由来間葉系幹細胞で見られる様な線維性組織形成、血管侵入や石灰化沈着は全く観察されなかった。これらの結果から、軟骨膜細胞はin vivoで生体組織と同様に軟骨実質部、軟骨膜部からなる組織構造を持つ弾性軟骨組織を長期的に再構築することが判明した。さらに、医療応用をめざすために同様の手法を用いてより大きな軟骨を再構築したところ、組織学的にも均一な細胞外基質を持っていることが判明した(図11a-b)。
In vivo tissue reconstruction capability of human perichondrocytes
We thought that a new elastic cartilage reconstruction method could be newly developed in vivo by using perichondrial cells that have high differentiation ability to chondrocytes and self-replication ability. To examine the feasibility of this approach, human perichondrial cells were transplanted subcutaneously in severely immunodeficient mice with viscous culture supernatant after induction of cartilage differentiation. For comparison, human chondrocytes were also transplanted in the same procedure. Perichondrial cells formed cartilage-like tissue 3 months after transplantation (FIG. 4i). Histological analysis revealed that perichondrocytes differentiate into mature chondrocytes in vivo, similar to chondrocytes, and reconstruct elastic cartilage tissues rich in proteoglycans and elastic fibers, which are their production substrates ( Figure 4i-p). On the other hand, immunohistochemical staining confirmed that Type I collagen (Col 1) positive membrane-like tissue covered the periphery of Col 2 positive elastic cartilage tissue only in tissues reconstructed from perichondrial cells. (Fig. 4p). In order to show that the reconstructed tissue was maintained over a long period of time, the same analysis was performed on tissues excised 6 months and 10 months after transplantation of perichondrial cells. At any time point, the excised tissue was covered with a Col 1 positive membrane-like tissue, and was found to be an elastic cartilage tissue composed of mature chondrocytes and their production matrix (FIGS. 10a-l). On the other hand, tumor formation, fibrous tissue formation, vascular invasion and calcification deposition as seen in bone marrow-derived mesenchymal stem cells were not observed at all. From these results, it has been found that perichondrial cells reconstruct long-term elastic cartilage tissue having a tissue structure composed of cartilage parenchyma and perichondrium as in vivo. Furthermore, when a larger cartilage was reconstructed using a similar technique for medical applications, it was found that it had a histologically uniform extracellular matrix (FIGS. 11a-b).
さらに、ヒト耳介軟骨から分離した軟骨膜細胞が、硝子軟骨組織の再構築能を有しているのか否かを検討することを目的として、重症免疫不全マウスの肋軟骨への移植実験を行った(Supplementary Methodsを参照)。移植後1ヶ月目の組織学的解析では、移植部に弾性線維を全く含まないヒト軟骨組織が再構築されていることが確認されたことから、ヒト軟骨膜細胞は弾性軟骨組織に加えて、硝子軟骨組織の再構築能を有していることが示唆された(図12)。 Furthermore, in order to investigate whether perichondrial cells isolated from human auricular cartilage have the ability to reconstitute hyaline cartilage tissue, we conducted transplantation experiments on severe cartilage of severely immunodeficient mice. (See Supplementary Methods). In histological analysis one month after transplantation, it was confirmed that the human cartilage tissue containing no elastic fibers at the transplanted part was reconstructed. It was suggested that it has the ability to reconstruct hyaline cartilage tissue (FIG. 12).
Elastic cartilage reconstruction using newly developed scaffold
ヒト軟骨膜細胞を用いた弾性軟骨組織の再構築に有益な新規スキャフォールド(pCol-HAp/ChS)の開発を行い、既存のスキャフォールドとの比較検討を行った。pCol-HAp/ChSは、コラーゲンスポンジに、軟骨基質として知られるコンドロイチン硫酸と力学的強度を上げるためにハイドロキシアパタイト粒子を付加(incorporate)することによって得た。力学的特性では高い圧縮弾性率を有しており、電子顕微鏡観察では、その内腔は連通性に富んでいた(図5a)。ヒト軟骨膜細胞を5mmX 5mm X 5mm大のpCol-HAp/ChSに播種した後、重症免疫不全マウスの皮下へ移植した(図6b)。既存のスキャフォールドと比較するために、同一条件下でハイドロキシアパタイトスキャフォールド(HAp)、コラーゲンスキャフォールド(Col)を用いた移植も実施した(図6e)。移植1ヶ月後、HApを用いた移植では、弾性軟骨組織は全く再構築されていなかった(Data not shown)。Colを用いた移植では、スキャフォールドの全体的な収縮がみられ、摘出した組織は摂子による用手圧迫で容易に扁平化した(図5 b-d)。スキャフォールド全域への細胞浸潤は認められず、組織学的にも基質産生は一部に限局していた(図5 b-d)。一方、pCol-HAp/ChSを用いた移植では、表面に光沢を持つ軟骨様組織を再構築した (図6, 5a,e-g,h-i)。再構築組織の硬さは耳介軟骨硬であり、用手圧迫後もその形態を留める弾力性を有していた(Supplementary movie)。Alcian Blue染色からは、軟骨基質産生を行う成熟軟骨細胞が、スキャフォールド全域にHomogenousに存在していることが明らかとなった(図5g)。そこで、より大型の弾性軟骨組織の再構築を目指し、直径15mm高さ20mmの円柱状pCol-HAp/ChSを用いて移植実験を行ったところ、1ヶ月後には12mm X 12mm X 20mm大の弾力を有するヒト軟骨様組織が再構築された (図5h)。組織学的解析により、スキャフォールド全域に成熟軟骨細胞が存在し、プロテオグリカン、弾性線維の産生が認められたことから、軟骨膜細胞はpCol-HAp/ChSと共に移植することでヒト弾性軟骨組織を再構築することが判明した(図5i)。
Elastic cartilage reconstruction using newly developed scaffold
We developed a new scaffold (pCol-HAp / ChS) useful for reconstructing elastic cartilage tissue using human perichondrial cells, and compared it with existing scaffolds. pCol-HAp / ChS was obtained by incorporating hydroxyapatite particles to chondroitin sulfate known as a cartilage matrix and mechanical strength to collagen sponge. In terms of mechanical properties, it has a high compressive elastic modulus, and its inner cavity was rich in communication through an electron microscope (FIG. 5a). Human perichondrial cells were seeded on 5 mm × 5 mm × 5 mm pCol-HAp / ChS and then transplanted subcutaneously into severely immunodeficient mice (FIG. 6 b). For comparison with the existing scaffold, transplantation using hydroxyapatite scaffold (HAp) and collagen scaffold (Col) was also performed under the same conditions (FIG. 6e). One month after transplantation, the elastic cartilage tissue was not reconstructed at all in the transplantation using HAp (Data not shown). In the transplantation using Col, the entire scaffold contraction was observed, and the excised tissue was easily flattened by manual compression with a pendulum (Fig. 5bd). Cell infiltration throughout the scaffold was not observed, and histologically, substrate production was limited to a part (Fig. 5bd). On the other hand, in transplantation using pCol-HAp / ChS, a cartilage-like tissue having a gloss on the surface was reconstructed (FIGS. 6, 5a, eg, hi). The hardness of the reconstructed tissue was auricular cartilage hardness, and it had the elasticity to retain its form after manual compression (Supplementary movie). Alcian Blue staining revealed that mature chondrocytes that produce cartilage matrix were present in homogenous throughout the scaffold (FIG. 5g). Therefore, aiming to reconstruct a larger elastic cartilage tissue, a transplantation experiment was performed using a cylindrical pCol-HAp / ChS with a diameter of 15 mm and a height of 20 mm. After one month, the elasticity of 12 mm x 12 mm x 20 mm was obtained. Having human cartilage-like tissue was reconstructed (FIG. 5h). Histological analysis revealed that mature chondrocytes were present throughout the scaffold, and proteoglycan and elastic fibers were produced. The perichondrial cells were transplanted with pCol-HAp / ChS to restore human elastic cartilage tissue. It was found to be constructed (Fig. 5i).
さらに我々は、軟骨膜細胞とpCol-HAp/ChSに三次元擬微小重力培養(図13b)を用いることによって、生体外で軟骨組織を再構築することを試みた(図13a)。軟骨膜細胞をpCol-HAp/ChS内に播種した後、擬微小重力培養装置下で40-50bpmにて培養することにより軟骨を再構築することが判明した(図13c)。 Furthermore, we attempted to reconstruct cartilage tissue in vitro by using three-dimensional pseudo-microgravity culture (Fig. 13b) for perichondrial cells and pCol-HAp / ChS (Fig. 13a). After seeding perichondrial cells in pCol-HAp / ChS, it was found that cartilage was reconstructed by culturing at 40-50 bpm in a pseudo-microgravity culture apparatus (FIG. 13c).
DISCUSSION
ヒト耳介軟骨組織における幹/前駆細胞の存在を明らかにすることは、耳介軟骨の発生過程や恒常性維持機構に対する理解を深めるだけでなく、弾性軟骨を対象とした質の高い再生治療を行う上で極めて重要である。我々は、ヒト耳介軟骨膜中に存在する、高い増殖能、軟骨・脂肪・骨分化能、自己複製能、組織再構築能を兼ね備えた幹/前駆細胞を初めて同定し、この細胞と新規開発した生分解性足場材料を用いることにより、ヒト弾性軟骨組織を長期的に再構築できることを明らかにした。我々が開発したヒト弾性軟骨再構築法は、次世代の弾性軟骨再生治療の実現にとって中核的な技術となる可能性がある。
DISCUSSION
Elucidating the presence of stem / progenitor cells in human auricular cartilage tissue not only deepens the understanding of the developmental process and homeostasis of auricular cartilage, but also provides high-quality regenerative treatment for elastic cartilage. It is extremely important to do. We identified for the first time a stem / progenitor cell that has high proliferative ability, cartilage / adipose / bone differentiation ability, self-replication ability, and tissue remodeling ability in human auricular perichondrium. It was clarified that human elastic cartilage tissue can be reconstructed in the long term by using the biodegradable scaffold material. The human elastic cartilage reconstruction method that we have developed may be a core technology for the realization of next-generation elastic cartilage regeneration therapy.
ヒト耳介などの弾性軟骨組織の臨床的再構築に使用可能な細胞源として、骨髄や脂肪組織から分離された間葉系幹細胞が重要であると考えられている。しかし、これらの間葉系幹細胞は、弾性軟骨に特徴的な細胞外マトリックスの構成成分であるプロテオグリカン、Col 2、エラスチン産生能を有した弾性軟骨細胞への分化能が極端に低いことが、臨床応用する上で極めて大きな障壁となっている14,15。さらに、骨髄由来間葉系幹細胞に関しては、生体内に移植した後に骨化や血管侵入などの問題が発生するリスクが高いことも臨床応用を阻む理由の一つとなっている17。本研究にて新たに同定されたヒト軟骨膜中に存在する幹/前駆細胞は、軟骨細胞への効率の良い分化能を有しており、従来から報告されている間葉系幹細胞とは明らかに性質が異なった、より軟骨細胞系列にコミットした幹/前細胞であることが推測された。一方、この細胞は、in vitroで骨・脂肪分化能を有していることや、in vivoで弾性線維を含まない硝子軟骨への分化能も兼ね備えていることから、弾性軟骨細胞系列へ完全に運命決定されている前駆細胞と比較すると、より上流に位置する細胞であると考えられる。実際、我々が新たに発見した幹/前駆細胞は、これまでに間葉系幹細胞の特異的マーカーとして報告されているCD44やCD90を共に発現しており、従来の間葉系幹細胞との類似性も認められているが、一方、CD133、CD140a、CD146、CD27127,29,30などの間葉系幹細胞マーカーは発現しておらず、それらとの相違性も存在している。よって、本幹/前駆細胞は、細胞系譜上、間葉系幹細胞に極めて近縁の位置にありながら、より軟骨細胞系列にコミットした軟骨幹/前駆細胞と呼ぶべき新たな細胞であると考えられる。現時点においては、間葉系細胞の分化系譜の詳細は不明であるが、今後の解析によりこれらが明らかになれば、本幹/前駆細胞の分化系譜上の位置づけが明確になるだろう。この様な理解が進むことにより、本幹/前駆細胞が弾性軟骨再生治療にとり最も優れた細胞源であることが明らかとなるだけでなく、硝子軟骨再生治療における細胞源としても有益であることが判明する可能性があり、膨大なニーズのある変形性関節症などに対しても臨床適応が拡大することが期待される。さらには、臨床応用の際に起こりうる有害事象、例えば、骨髄由来間葉系幹細胞で問題になっている骨組織の形成、などのリスク発生率の推測を行うための一助となるであろう。 Mesenchymal stem cells isolated from bone marrow and adipose tissue are considered important as a cell source that can be used for clinical reconstruction of elastic cartilage tissues such as human auricles. However, these mesenchymal stem cells have an extremely low ability to differentiate into elastic chondrocytes capable of producing proteoglycan, Col 2 and elastin, which are components of the extracellular matrix characteristic of elastic cartilage. 14 and 15 and has a very large barrier in terms of application. In addition, bone marrow-derived mesenchymal stem cells are one of the reasons for hindering clinical application because of the high risk of occurrence of ossification and vascular invasion after transplantation in vivo 17 . Stem / progenitor cells present in human perichondrium newly identified in this study have an efficient differentiation ability into chondrocytes, and are clearly different from previously reported mesenchymal stem cells It was speculated that this is a stem / progenitor cell committed to a more chondrocyte lineage with different properties. On the other hand, these cells have the ability to differentiate into bone and fat in vitro, and also have the ability to differentiate into hyaline cartilage that does not contain elastic fibers in vivo. Compared with the progenitor cells whose fate has been determined, the cells are considered to be located upstream. In fact, the newly discovered stem / progenitor cells express both CD44 and CD90, which have been reported as specific markers for mesenchymal stem cells, and are similar to conventional mesenchymal stem cells. On the other hand, mesenchymal stem cell markers such as CD133, CD140a, CD146, CD271 27 , 29, and 30 are not expressed, and there are also differences. Therefore, this stem / progenitor cell is considered to be a new cell that should be called a cartilage stem / progenitor cell committed to a more chondrocyte lineage while being in a position closely related to the mesenchymal stem cell on the cell lineage. . At present, the details of the differentiation lineage of mesenchymal cells are unknown, but if these are revealed by future analysis, the position of the stem / progenitor cells in the differentiation lineage will be clear. This understanding will not only reveal that stem / progenitor cells are the most excellent cell source for elastic cartilage regeneration therapy, but may also be useful as a cell source for hyaline cartilage regeneration therapy. There is a possibility that it will become clear, and it is expected that clinical indications will be expanded even for osteoarthritis and the like that have enormous needs. Furthermore, it will help to estimate the incidence of risks such as adverse events that can occur during clinical application, such as the formation of bone tissue that is a problem with bone marrow-derived mesenchymal stem cells.
造血幹細胞や角膜上皮幹細胞などの組織幹細胞を利用した再生医療は、幹細胞の持つ自己複製能により成熟細胞が長期間に渡り継続的に供給されることから、臨床的に優れた組織再構築法となることが期待されている31。我々は、本幹/前駆細胞を積層化培養による軟骨分化誘導後に産生基質と共に皮下移植する方法により、in vivoにおいてヒト弾性軟骨組織が再構築される事を見いだした。再構築されたヒト弾性軟骨組織は、生体組織と同様に軟骨膜部、軟骨実質部からなる組織構造をとる事が明らかとなった。したがって、再構築された軟骨膜中に存在する幹/前駆細胞の自己複製による不断の細胞更新と軟骨細胞分化が期待されるため、優れた組織維持性を有していることが大いに期待される。実際、移植6、10ヶ月後の解析においても、再構築された弾性軟骨組織は完全に維持されており、長期形態維持性を有していることが確認されている。また、線維性組織形成、血管侵入や石灰化沈着は10ヶ月の間で全く見られなかったことから、本幹/前駆細胞は弾性軟骨再生治療の実現にとり骨髄由来間葉系幹細胞よりも優れた細胞源であることが推測される。小児先天奇形を対象とした弾性軟骨再生治療では、患児の一生涯に渡り軟骨組織が恒常的に維持されることが必須であるため、Serial transplantation法などを利用して、本幹/前駆細胞の自己複製能の臨界点を明らかにしていく必要があるだろう。 Regenerative medicine using tissue stem cells such as hematopoietic stem cells and corneal epithelial stem cells is a clinically superior tissue reconstruction method because mature cells are continuously supplied over a long period of time due to the self-replicating ability of stem cells. 31 is expected to be. We have found that human elastic cartilage tissue is reconstructed in vivo by a method in which the stem / progenitor cells are transplanted subcutaneously together with the production matrix after induction of cartilage differentiation by layered culture. It was clarified that the reconstructed human elastic cartilage tissue has a tissue structure composed of a perichondrium portion and a cartilage parenchymal portion, like a living tissue. Therefore, continuous cell renewal and chondrocyte differentiation due to self-replication of stem / progenitor cells present in the reconstructed perichondrium are expected, so it is highly expected to have excellent tissue maintenance . In fact, it was confirmed that the reconstructed elastic cartilage tissue was completely maintained in the analysis 6 and 10 months after transplantation, and had long-term morphology maintenance. In addition, since no fibrous tissue formation, vascular invasion or calcification deposition was observed in 10 months, the stem / progenitor cells were superior to the bone marrow-derived mesenchymal stem cells for realizing elastic cartilage regeneration therapy Presumed to be a cell source. In elastic cartilage regeneration treatment for congenital malformations in children, it is essential that the cartilage tissue is constantly maintained throughout the lifetime of the patient. It will be necessary to clarify the critical point of self-replicating ability.
ヒト弾性軟骨の再生治療を広く実現化するためには、優れた細胞源に加え形態制御を可能とする足場材料が必要不可欠である。実際、無細胞化された同種軟骨組織を足場材料として、ヒト気管軟骨の再構築を試みる臨床試験が実施され、良好な成果が報告されている32。高品質の足場材料は組織再構築法の臨床応用にとり極めて重要な要素技術のひとつであり、連通性、高圧縮弾性率、良好な生分解性を併せ持つヒト弾性軟骨の再構築に有益な革新的足場材料の開発が急務である。我々は、コラーゲンスポンジにコンドロイチン硫酸とハイドロキシアパタイト粒子を付加することで優れた力学的特性を有する画期的な生分解性足場材料を開発した。驚くべきことに、再構築組織中には、豊富な弾性線維、プロテオグリカン産生を行う成熟軟骨細胞がHomogenousに局在していることが明らかとなった。本スキャフォールド中に軟骨組織における細胞外マトリックスの主要な構成成分であるコラーゲンとコンドロイチン硫酸を含有させていることが、軟骨幹/前駆細胞の吸着・浸潤性や増殖・分化に有利な細胞外環境を提供しているものと考えられた33,34。 In order to widely realize regenerative treatment of human elastic cartilage, a scaffold material capable of controlling morphology in addition to an excellent cell source is indispensable. In fact, as acellularized allogeneic cartilage tissue scaffold material, it is carried out clinical trials to try to reconstruct the human tracheal cartilage, and good results have been reported 32. High-quality scaffolding material is one of the most important elemental technologies for clinical application of tissue remodeling method, and it is an innovative that is beneficial for reconstructing human elastic cartilage that has both connectivity, high compressive modulus and good biodegradability There is an urgent need to develop scaffold materials. We have developed an innovative biodegradable scaffold material with excellent mechanical properties by adding chondroitin sulfate and hydroxyapatite particles to collagen sponge. Surprisingly, it became clear that mature chondrocytes that produce abundant elastic fibers and proteoglycans are localized in homogenous in the reconstructed tissue. Extracellular environment advantageous for cartilage stem / progenitor adsorption / invasion and proliferation / differentiation by including collagen and chondroitin sulfate, which are the main components of extracellular matrix in cartilage tissue, in this scaffold 33,34 thought to have provided.
従来、臨床ニーズに十分応えることができる優れた弾性軟骨再構築法は存在しなかった。唯一、臨床応用例のある耳介軟骨細胞を用いた方法でも、成熟軟骨細胞を用いているが故の組織吸収と細胞移植であるための適応限定が解決課題として残されており、小児期の先天奇形などへの臨床適応の拡大を阻んできた35。しかし、我々は低侵襲操作で採取可能なヒト耳介軟骨膜から幹/前駆細胞を分離し、培養系を用いて分化誘導後、細胞を皮下注入する、という極めて簡便な細胞操作技術による弾性軟骨組織の再構築法を開発した。本法を用いれば、従来法における課題を克服できるばかりか、成長に応じた追加注入も容易に可能であるため、単純な形状の小組織欠損に基づく小児先天奇形に対する治療戦略が確立されたといえるだろう。さらに、我々は耳介軟骨のような複雑な構造を有する大型の弾性軟骨組織を再構築するために不可欠となる足場材料の開発を行い、本幹/前駆細胞と組み合わせることによって弾性軟骨組織を再構築させることに成功した。今後、本足場材料の臨床応用の可能性が検討され、耳介軟骨などの大型で複雑な形態に成形するための技術開発がなされれば、形成外科・美容外科領域における多種多様な臨床ニーズに合わせた弾性軟骨組織の再構築を行う上で中核技術となることが大いに期待される。 Heretofore, there has been no excellent elastic cartilage reconstruction method that can sufficiently meet clinical needs. The only method using auricular chondrocytes with clinical applications is that the limited use of tissue resorption and cell transplantation due to the use of mature chondrocytes remains a problem to be solved. 35 This has hindered the expansion of clinical indications for congenital malformations. However, we have isolated the stem / progenitor cells from human auricular perichondrium that can be collected by a minimally invasive procedure, induced differentiation using a culture system, and then injected the cells subcutaneously. A restructuring method of the organization was developed. By using this method, not only can the problems of the conventional method be overcome, but it is also possible to easily perform additional injection according to growth, so it can be said that a treatment strategy for congenital malformations of children based on small tissue defects of simple shape has been established. right. Furthermore, we have developed a scaffold material that is indispensable for reconstructing large elastic cartilage tissues with complex structures such as auricular cartilage, and recombining elastic cartilage tissues by combining with main stem / progenitor cells. Successfully built. In the future, the possibility of clinical application of this scaffold material will be studied, and if technical development for molding into large and complex shapes such as auricular cartilage is made, it will meet a wide variety of clinical needs in the field of plastic surgery and cosmetic surgery. It is highly expected to become a core technology for reconstructing the combined elastic cartilage tissue.
Methods
Isolation and cultivation of human perichondrocytes
横浜市立大学附属病院倫理委員会より承認を得て(approval #03-074)、30人の小耳症患者より、手術の際に余剰となる残存耳介弾性軟骨を供与頂き研究を遂行した。実体顕微鏡下で軟骨膜部、軟骨膜-軟骨移行部、軟骨実質部の3層に分離し、組織を細切後、0.2%コラゲナーゼタイプII（Worthington）に懸濁・振蕩した。各組織の細胞懸濁液は100μm ナイロンメッシュ（BD Biosciences）で濾過し、PBSによる洗浄を3度行った。細胞懸濁液は、37 ℃、CO2 5 %に設定したインキュベータで10 % fetal bovine serum (MOREGATE)、1 % Antibiotic Antimycotic Solution（SIGMA）を含有するDulbecco’s modified Eagle medium and Ham’s F-12 medium（日水製薬）を含む増殖培地により培養を行った。尚、長期増殖能の評価では、継代に際し血球計算板を用いて細胞数をカウントした後、1200 cells/cm2の密度で35mmディッシュに播種し、コンフルエントに達した際に同様に継代をする、という操作を繰り返した。
Methods
Isolation and cultivation of human perichondrocytes
Obtained approval from the Yokohama City University Hospital Ethics Committee (approval # 03-074), we conducted a study by providing surplus residual auricular elastic cartilage at the time of surgery from 30 patients with microtia. Under a stereomicroscope, it was separated into three layers of perichondrium, perichondrium-cartilage transition, and cartilage parenchyma, and the tissue was minced and suspended and shaken in 0.2% collagenase type II (Worthington). The cell suspension of each tissue was filtered through a 100 μm nylon mesh (BD Biosciences) and washed with PBS three times. The cell suspension was Dulbecco's modified Eagle medium and Ham's F-12 medium (day) containing 10% fetal bovine serum (MOREGATE) and 1% Antibiotic Antimycotic Solution (SIGMA) in an incubator set at 37 ° C and 5% CO 2. Cultivation was carried out in a growth medium containing Mizuho Pharmaceutical. In the evaluation of long-term proliferative capacity, the number of cells was counted using a hemocytometer at the time of passage, and then seeded in a 35 mm dish at a density of 1200 cells / cm 2 , and passage was similarly performed when the cells reached confluence. Repeated the operation.
In vitro colony assay
各細胞を、35 mm細胞培養ディッシュに52 cells/cm2の密度で播種した。14日間の増殖培地による培養後、コロニー数のカウントを行った。カウントに際してはギムザ染色（武藤化学薬品）による染色を行った後、50個以上の細胞集団を1コロニーとし定量した。
In vitro colony assay
Each cell was seeded at a density of 52 cells / cm 2 in a 35 mm cell culture dish. After culturing for 14 days in the growth medium, the number of colonies was counted. In counting, after staining with Giemsa staining (Muto Chemical), 50 or more cell populations were quantified as one colony.
FACS analysis and cell sorting
軟骨膜細胞と軟骨細胞は第４継代培養の後、各々1×106細胞毎に1 mgのFluorescein isothiocyanate(FITC)、 Phycoerythrin(PE) 、 Allophycocyanin(APC)抱合モノクローナル抗体で4℃、30分間染色した. 3度の洗浄後、1μg/mlのpropidium iodide(PI) を含むPBSに懸濁し、 FACSによる解析を行った.解析にはMoFlo cell sorter (DakoCytomation)を用い、染色には 抗CD28、 CD34、 CD44、 CD49e、 CD73、 CD90、 CD105、CD117(c-kit)、 CD133、 CD138、 CD140a、 CD146、 CD271抗体を用いた。軟骨膜細胞に対し、抗CD44、CD90抗体を用いてソーティングを行った。ソーティングゲートは、CD44+ CD90+、CD44+ CD90‐、CD44‐ CD90+、CD44‐ CD90‐に対して設定し、各分画の細胞は６穴ディッシュ（BD Biosciences）に52 cells/cm2の密度で播種した。ソーティングに際し、細胞の残骸、死細胞やダブレットは前方散乱光、側方散乱光、PIによって除去された。
FACS analysis and cell sorting
Perichondrial cells and chondrocytes are subcultured at 4 ° C for 30 min with 1 mg of Fluorescein isothiocyanate (FITC), Phycoerythrin (PE) and Allophycocyanin (APC) conjugated monoclonal antibodies after every 4 × 4 cells. After washing 3 times, the suspension was suspended in PBS containing 1 μg / ml propidium iodide (PI) and analyzed by FACS.MoFlo cell sorter (DakoCytomation) was used for the analysis, and anti-CD28, CD34, CD44, CD49e, CD73, CD90, CD105, CD117 (c-kit), CD133, CD138, CD140a, CD146, and CD271 antibodies were used. Sorting was performed on perichondrocytes using anti-CD44 and CD90 antibodies. Sorting gate, CD44 + CD90 +, CD44 + CD90-, CD44- CD90 +, CD44- set for CD90-, each fraction of the cells was seeded at a density of 52 cells / cm 2 in 6-well dishes (BD Biosciences). During sorting, cell debris, dead cells and doublets were removed by forward scattered light, side scattered light and PI.
Multipotent differentiation in vitro
軟骨分化誘導に関しては積層化培養法を用いた。各細胞を2.5×104 cells/cm2の密度で播種し、播種後48時間まで増殖培地で培養を行い、その後はL-ascorobic acid 2-phosphate（SIGMA）、Dexamethasone（SIGMA）、human-recombinant Insulin-like Growth Factor-I（SIGMA）、human-recombinant basic Fibroblast Growth Factor（Wako）を添加した分化誘導培地を用いて培養を行った。5日間の培養の後、さらに2.5×104 cells/cm2の細胞を上に播種し、同様の手順で培養を行うという操作を計2回繰り返した。骨および脂肪分化誘導に関しては、以前の報告に準じた36。
Multipotent differentiation in vitro
For induction of cartilage differentiation, the layered culture method was used. Each cell is seeded at a density of 2.5 × 10 4 cells / cm 2 and cultured in a growth medium for 48 hours after seeding, and then L-ascorobic acid 2-phosphate (SIGMA), Dexamethasone (SIGMA), human-recombinant Culturing was performed using a differentiation induction medium supplemented with insulin-like growth factor-I (SIGMA) and human-recombinant basic fibroblast growth factor (Wako). After culturing for 5 days, an operation of further seeding 2.5 × 10 4 cells / cm 2 of cells and culturing in the same procedure was repeated twice in total. As for the induction of bone and adipose differentiation, 36 according to previous reports.
Gene expression analysis
軟骨・脂肪・骨分化能を調べるため、Reverse-transcription
polymerase chain reaction (RT-PCR) 、並びにquantitative PCR (qPCR)を行った。RT-PCRによる各分化関連遺伝子の発現の確認には以下のプライマーを用いた。type I collagen(COL1A1)、type II collagen(COL2A1)、type X collagen(COL10A1)、aggrecan(ACAN)、elastin(ELN)、lipoprotein lipase(Lpl/LPL)、C/EBPα(Cebpa/CEBPA)、aP2(Fabp4)、Adipsin(CFD) 、PPARγ PPARGC1A)、runt-related transcription factor 2 (Runx2) 、alkaline phosphatase (ALPL)を用いた(Supplementary methods online)。qPCRには、TaqMan（登録商標）Gene Expression Assays（Applied Biosystems）のCOL1A1：Hs00266273_ml、COL2A1：Hs00164099_ml、CSPG2：Hs01007933_m1、ELN：Hs00355783_ml、FBN1：Hs00171191_m1 のprimer/probe setを用いた。
Gene expression analysis
Reverse-transcription to examine cartilage, fat and bone differentiation potential
Polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) were performed. The following primers were used to confirm the expression of each differentiation-related gene by RT-PCR. type I collagen (COL1A1), type II collagen (COL2A1), type X collagen (COL10A1), aggrecan (ACAN), elastin (ELN), lipoprotein lipase (Lpl / LPL), C / EBPα (Cebpa / CEBPA), aP2 ( Fabp4), Adipsin (CFD), PPARγ PPARGC1A), runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALPL) were used (Supplementary methods online). For qPCR, a primer / probe set of TaqMan (registered trademark) Gene Expression Assays (Applied Biosystems) COL1A1: Hs00266273_ml, COL2A1: Hs00164099_ml, CSPG2: Hs01007933_m1, ELN: Hs00355783_ml, FBN1: Hs00171191_m1 was used.
ELISA
積層化培養による軟骨分化誘導下で培養1、３週間目に、培養上清を回収した。上清中に分泌されたプロテオグリカン、エラスチン、コラーゲンを、それぞれBLYSCAN、FASTIN、SIRCOL assays(Biocolor)のキットを用いて定量測定を行った37。
ELISA
The culture supernatant was collected at 1 and 3 weeks of culture under induction of cartilage differentiation by layered culture. Proteoglycan secreted into the supernatant, elastin, collagen, were respectively subjected Blyscan, FASTIN, a quantitative measurement using the kit SIRCOL assays (Biocolor) 37.
In vivo transplantation
軟骨分化誘導を行った細胞をその産生基質とともにシリンジに回収し、重症免疫不全マウス(NOD/SCID)(三協)の背部皮下に1 mlずつ移植を行った。Scaffoldに細胞を播種した群では、円柱上にくり抜いたpCol-HAp/ChS(Tokyo Institute of Technology)、Collagen sponge(テルモ)、ハイドロキシアパタイトスキャフォールド(オリンパス)に各々3カ所ずつ計1mlの細胞浮遊液を添加し、NOD/SCIDマウスの背部皮下に移植した。移植後、4週目、12週及び40週目に摘出を行い、組織学的に検討した。尚、実験動物の飼育、取り扱いに関しては横浜市立大学医学部動物実験センターの規定に基づき行った。
In vivo transplantation
Cells subjected to cartilage differentiation induction were collected in a syringe together with the production substrate, and 1 ml each was transplanted subcutaneously in the back of severely immunodeficient mice (NOD / SCID) (Sankyo). In the group in which cells were seeded on Scaffold, 3 ml each of pCol-HAp / ChS (Tokyo Institute of Technology), Collagen sponge (Terumo), and hydroxyapatite scaffold (Olympus) hollowed out on a cylinder, totaling 1 ml of cell suspension. And was implanted subcutaneously into the back of NOD / SCID mice. After transplantation, excision was performed at 4, 12, and 40 weeks, and histological examination was performed. The breeding and handling of experimental animals was conducted according to the regulations of the Yokohama City University School of Medicine Animal Experiment Center.
Methods for newly invented scaffolds
pCol-HAp/ChSスキャホールドの製造のための詳細な方法は、以下の論文に記載されている。
Ohyabu Y.,et al. A Collagen Sponge Incorporating a Hydroxyapatite/Chondroitinsulfate Composite as a Scaffold for Cartilage Tissue Engineering. J of Biomaterials Science 20,1861-1874 (2009)
Adegawa T.,et al. Preparation and Characterization of Porous Scaffolds Consisted of Hydroxyapatite, Polysaccharides and Collagen for Cartilage Tissue Engineering.Key Engineering Materials 396-398, 707-710 (2009)
Methods for newly invented scaffolds
Detailed methods for the production of pCol-HAp / ChS scaffolds are described in the following papers.
Ohyabu Y., et al. A Collagen Sponge Incorporating a Hydroxyapatite / Chondroitinsulfate Composite as a Scaffold for Cartilage Tissue Engineering.J of Biomaterials Science 20,1861-1874 (2009)
Adegawa T., et al. Preparation and Characterization of Porous Scaffolds Consisted of Hydroxyapatite, Polysaccharides and Collagen for Cartilage Tissue Engineering.Key Engineering Materials 396-398, 707-710 (2009)
Histochemical and immunohistochemical analysis
組織切片、または培養細胞を固定した後、H&E、アルシアンブルー、エラスチカ・ワンギーソン、アリザリンレッドS、オイルレッドO（武藤化学薬品）で組織化学染色を行った。免疫組織化学染色に際しては、rabbit anti-human type I collagen monoclonal antibody(Col1)（MONOSAN）、mouse anti-chicken type II collagen polyclonal antibody(Col2)（CHEMICON）を用いて4 ℃、over nightで反応させた。洗浄後、適切な動物種に対するAlexa488- and/or Cy3-conjugated二次抗体（1:800、Molecular Probes）を添加し、室温で1時間反応させた。その後4’,6-diamidino-2-phenylindole (DAPI) を添加したFA Mounting Fluid（BD Biosciences）にて核染色及び封入を行い、LSM510 Laser Scanning Microscope（ZEISS）を用いて観察、画像を撮影した。
Histochemical and immunohistochemical analysis
After fixing tissue sections or cultured cells, histochemical staining was performed with H & E, Alcian Blue, Elastica Wangyson, Alizarin Red S, Oil Red O (Mudo Chemicals). For immunohistochemical staining, the reaction was carried out at 4 ° C over night using rabbit anti-human type I collagen monoclonal antibody (Col1) (MONOSAN) and mouse anti-chicken type II collagen polyclonal antibody (Col2) (CHEMICON). . After washing, an Alexa488- and / or Cy3-conjugated secondary antibody (1: 800, Molecular Probes) against an appropriate animal species was added and allowed to react at room temperature for 1 hour. Thereafter, nuclear staining and encapsulation were performed with FA Mounting Fluid (BD Biosciences) to which 4 ′, 6-diamidino-2-phenylindole (DAPI) was added, and observation and images were taken using an LSM510 Laser Scanning Microscope (ZEISS).
Statistical analysis
データは、少なくとも3人以上の独立した検体による実験から得たmean±s.d.を表記した。統計学的解析には、まず3あるいは4群のデータに対しKruskal Wallis-H testを行い、P＜0.01と判定された場合に、Mann-Whitney’s U test with Bonferroni correctionによる多重比較検定を行った。有意確率P値がP＜0.001またはP＜0.01を満たす場合を統計学的有意差ありと判定した。
Statistical analysis
Data are expressed as mean ± sd obtained from experiments with at least 3 independent specimens. For statistical analysis, the Kruskal Wallis-H test was first performed on the data in groups 3 and 4, and when P <0.01, a multiple comparison test by Mann-Whitney's U test with Bonferroni correction was performed. When the significance probability P value satisfied P <0.001 or P <0.01, it was determined that there was a statistically significant difference.
Supplementary Methods
Label-retaining cells
エーテル（昭和エーテル Japan）で麻酔した妊娠17.5日目の母体マウスにBrdU（50μg/g body WT)(Sigma-Aldrich, St Louis, MO, USA)を12時間毎、計6回腹腔内投与した。生まれた仔の耳介を各週齢（新生仔〜2週齢、4週齢、24週齢、48週齢）の段階において4% paraformaldehydeで固定して解析に用いた。
Supplementary Methods
Label-retaining cells
BrdU (50 μg / g body WT) (Sigma-Aldrich, St Louis, MO, USA) was intraperitoneally administered 6 times every 12 hours to a 17.5 day pregnant mother anesthetized with ether (Showa Ether Japan). The pinna of the born pups were fixed with 4% paraformaldehyde and used for analysis at the stage of each week (newborn to 2 weeks old, 4 weeks old, 24 weeks old, 48 weeks old).
Immunohistochemistry
組織切片の作製と免疫組織化学染色は賦活化を省いた以外はBrdUと同様に行った。用いた一次抗体と二次抗体の希釈倍率は、以下の通りである。
α1-integrin(hamstar,1:100,BD Biosciences Sun Jose, CA USA),α2-integrin(hamstar,1:100,BD Biosciences),α5-integrin(rabbit,1:100,Chemicon,Temecula,CA USA),α6-integrin(rat,1:100,Chemicon),αV-integrin(rabbit,1:100,Chemicon）,αL-integrin(rat,1:100,eBioscience,San Diego,CA USA),αM-integrin antibody(rat,1:100,eBioscience),αX-integrin (hamster,1:100,eBioscience),β1-integrin(hamster,1:100,BD Biosciences),β2-integrin(rat,1:100,eBiosciences),β3-integrin(hamster,1:100,BD
Biosciences), CD44 (rat,1:100,Chemicon), Syndecan-1 (rat, 1:100,BD
Biosciences),Syndecan-3(rabbit,1:100, Santa Cruz),Syndecan-4 (rat,1:100,BD
Biosciences ), PECAM(rat,1:100,BD Biosciences), VCAM-1(rat, 1:100,Chemicon）, Flk-1(rat, 1:100,BD Biosciences) Cy3-conjugated Donkey anti sheep IgG antibody（1:1600, Chemicon）, Alexa488-conjugated Donkey anti sheep IgG antibody（1:1200, Molecular Probes,CA, USA）,Cy3-conjugated Donkey anti Rabbit IgG antibody（1:1600, Jackson Immuno Research LABORATORIES, INC.West Grove,PA,USA）
Immunohistochemistry
Preparation of tissue sections and immunohistochemical staining were performed in the same manner as BrdU except that activation was omitted. The dilution ratio of the used primary antibody and secondary antibody is as follows.
α 1 -integrin (hamstar, 1: 100, BD Biosciences Sun Jose, CA USA), α 2 -integrin (hamstar, 1: 100, BD Biosciences), α 5 -integrin (rabbit, 1: 100, Chemicon, Temecula, CA USA), α 6 -integrin (rat, 1: 100, Chemicon), α V -integrin (rabbit, 1: 100, Chemicon), α L -integrin (rat, 1: 100, eBioscience, San Diego, CA USA ), α M -integrin antibody (rat, 1: 100, eBioscience), α X -integrin (hamster, 1: 100, eBioscience), β 1 -integrin (hamster, 1: 100, BD Biosciences), β 2 -integrin (rat, 1: 100, eBiosciences), β 3 -integrin (hamster, 1: 100, BD
Biosciences), CD44 (rat, 1: 100, Chemicon), Syndecan-1 (rat, 1: 100, BD
Biosciences), Syndecan-3 (rabbit, 1: 100, Santa Cruz), Syndecan-4 (rat, 1: 100, BD
Biosciences), PECAM (rat, 1: 100, BD Biosciences), VCAM-1 (rat, 1: 100, Chemicon), Flk-1 (rat, 1: 100, BD Biosciences) Cy3-conjugated Donkey anti sheep IgG antibody ( 1: 1600, Chemicon), Alexa488-conjugated Donkey anti sheep IgG antibody (1: 1200, Molecular Probes, CA, USA), Cy3-conjugated Donkey anti Rabbit IgG antibody (1: 1600, Jackson Immuno Research LABORATORIES, INC. West Grove , PA, USA)
Long-term cell growth assay
細胞の増殖能を解析するために、各細胞を、35 mmイージーグリップ細胞培養ディッシュに（FALCON）1200 cells/cm2播種した。各細胞は10 %ウシ胎児血清（MOREGAE）、1 % Antibiotic Antimycotic Solution（SIGMA）を添加したDulbecco’s modified Eagle medium and Ham’s F-12 medium（日水製薬）を用い、気相条件を37 ℃、CO2濃度5 %に設定したインキュベータ内で培養を行った。培地交換は播種24時間後から３日に1回行った。14日間の培養後、0.2 %タイプIIコラゲナーゼ溶液を用いて細胞を剥離し、血球計算板を用いて細胞数を計測した後、再び35 mmイージーグリップ細胞培養ディッシュ（FALCON）に1200 cells/cm2播種した。この操作を繰り返して行い、長期における細胞の増殖能維持能を調べた。
Long-term cell growth assay
In order to analyze the proliferation ability of the cells, each cell was seeded in a 35 mm Easy Grip cell culture dish (FALCON) 1200 cells / cm 2 . Each cell 10% fetal calf serum (MOREGAE), with 1% Antibiotic Antimycotic Dulbecco was added Solution (SIGMA)'s modified Eagle medium and Ham's F-12 medium ( Nissui Pharmaceutical), 37 ° C. The vapor phase conditions, CO 2 Culturing was performed in an incubator set at a concentration of 5%. The medium was exchanged once every 3 days from 24 hours after sowing. After culturing for 14 days, the cells were detached using a 0.2% type II collagenase solution, the number of cells was counted using a hemocytometer, and then again on a 35 mm easy grip cell culture dish (FALCON) at 1200 cells / cm 2 Sowing. This operation was repeated to examine the ability to maintain cell growth ability over a long period of time.
In vitro colony assay.
To assess colony formation, we cultured non-sorted or sorted cells in our standard culture medium at a density of 52 cells/cm2 under an atmosphere of 5% CO2 at 37°C. The culture medium was replaced every 7 days. The colonies were stained with Giemsa and counted 14 or 21 days after seeding.
クローン性コロニーの形成能を解析するために、各細胞を35 mmイージーグリップ細胞培養ディッシュ（FALCON）に52 cells/cm2播種した。各細胞は10 %ウシ胎児血清（MOREGAE）、1 % Antibiotic Antimycotic Solution（SIGMA）を添加したDulbecco’s modified Eagle medium and Ham’s F-12 medium（日水製薬）を用い、気相条件を37 ℃、CO2濃度5 %に設定したインキュベータ内で培養を行った。培地交換は播種24時間後から３日に1回行った。14日間の培養後、ギムザ染色を行い、計測した。
In vitro colony assay.
To assess colony formation, we cultured non-sorted or sorted cells in our standard culture medium at a density of 52 cells / cm2 under an atmosphere of 5% CO2 at 37 ° C. The culture medium was replaced every 7 days. stained with Giemsa and counted 14 or 21 days after seeding.
In order to analyze the ability to form clonal colonies, each cell was seeded on a 35 mm easy grip cell culture dish (FALCON) at 52 cells / cm 2 . Each cell 10% fetal calf serum (MOREGAE), with 1% Antibiotic Antimycotic Dulbecco was added Solution (SIGMA)'s modified Eagle medium and Ham's F-12 medium ( Nissui Pharmaceutical), 37 ° C. The vapor phase conditions, CO 2 Culturing was performed in an incubator set at a concentration of 5%. The medium was exchanged once every 3 days from 24 hours after sowing. After 14 days of culture, Giemsa staining was performed and counted.
RT-PCR
軟骨膜細胞と軟骨細胞からRNeasy
(QIAGEN)によりTotal RNAを抽出した。cDNA は RNA PCR kit (Takara)により得られた。プライマーはPrimer 3により下記のようにデザインした。軟骨分化マーカーとしてtype I collagen(COL1A1) ; forward 5’cgacagaggcataaagggtca3’（配列番号１）,reverse 5’ tacacgcaggtctcaccagtctc3’ （配列番号２） ; type II collagen(COL2A1); forward 5 ctggctcccaacactgccaacgtc3’ （配列番号３）, reverse 5’ tcctttgggtttgcaacggattgt3’ （配列番号４） ;type X collagen(COL10A1); forward 5’ cccactacccaacaccaagac3’ （配列番号５）, reverse 5’ tttctgtccattcataccaggg3’ （配列番号６）;aggrecan(ACAN); forward 5’gtatgtgaggagggctggaaca3’ （配列番号７）, reverse
5’cgcttctgtagtctgcgtttgta3’ （配列番号８）;elastin(ELN); forward 5’ tatggactgccctacaccacag3’ （配列番号９）, reverse 5’ agcacctgggacaactggaat3’ （配列番号１０）を用いた。脂肪分化マーカーとしてlipoprotein lipase(Lpl/LPL);forward 5’ tggacggtaacaggaatgtatgag3’ （配列番号１１）, reverse 5’ ccctctggtgaatgtgtgtaaga3’ （配列番号１２）; aP2(Fabp4); forward 5’ ggtacctggaaacttgtctccag3’ （配列番号１３）, reverse 5’ catgacgcattccaccaccag3’ （配列番号１４） ; PPARγ(PPARGC1A); forward 5’ gtgtgctgctctggttggtgaagac3’, （配列番号１５） reverse 5’ gttggctggtgccagtaagagcttc3’ （配列番号１６）
を用いた。骨分化マーカーとしてrunt-related transcription factor 2 (Runx2) ; forward 5’ gagtttcaccttgaccataaccgtcttcac3’ （配列番号１７）, reverse 5’gtggtagagtggatggacgggg3’ （配列番号１８）;alkaline phosphatase (ALPL) ; forward 5’
tcccggtgcaacaccacccag3’ （配列番号１９）, reverse 5’ caacgaggtccaggccgtcc3’ （配列番号２０）を用いた。
RT-PCR
RNeasy from perichondrial cells and chondrocytes
Total RNA was extracted by (QIAGEN). cDNA was obtained with RNA PCR kit (Takara). Primers were designed by Primer 3 as follows. Type I collagen (COL1A1); forward 5'cgacagaggcataaagggtca3 '(SEQ ID NO: 1), reverse 5' tacacgcaggtctcaccagtctc3 '(SEQ ID NO: 2); type II collagen (COL2A1); forward 5 ctggctcccaacactgccaacgtc3' (SEQ ID NO: 3) reverse 5 'tcctttgggtttgcaacggattgt3' (sequence number 4); type X collagen (COL10A1); forward 5 'cccactacccaacaccaagac3' (sequence number 5), reverse 5 'tttctgtccattcataccaggg3' (sequence number 6); aggrecan (ACAN); forward 5'gtatgtgaggaggggggg (SEQ ID NO: 7), reverse
5'cgcttctgtagtctgcgtttgta3 '(SEQ ID NO: 8); elastin (ELN); forward 5' tatggactgccctacaccacag3 '(SEQ ID NO: 9), reverse 5' agcacctgggacaactggaat3 '(SEQ ID NO: 10) was used. Lipoprotein lipase (Lpl / LPL); forward 5 'tggacggtaacaggaatgtatgag3' (SEQ ID NO: 11), reverse 5 'ccctctggtgaatgtgtgtaaga3' (SEQ ID NO: 12); aP2 (Fabp4); forward 5 'ggtacctggaaacttgtctccag3' (SEQ ID NO: 13) reverse 5 'catgacgcattccaccaccagag' (SEQ ID NO: 14); PPARγ (PPARGC1A); forward 5 'gtgtgctgctctggttggtgaagac3', (SEQ ID NO: 15) reverse 5 'gttggctggtgccagtaagagcttc3' (SEQ ID NO: 16)
Was used. Runt-related transcription factor 2 (Runx2); forward 5 'gagtttcaccttgaccataaccgtcttcac3' (SEQ ID NO: 17), reverse 5'gtggtagagtggatggacgggg3 '(SEQ ID NO: 18); alkaline phosphatase (ALPL); forward 5'
tcccggtgcaacaccacccag3 ′ (SEQ ID NO: 19) and reverse 5 ′ caacgaggtccaggccgtcc3 ′ (SEQ ID NO: 20) were used.
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1. Chang, SC, Tobias, G., Roy, AK, Vacanti, CA & Bonassar, LJ Tissue engineering of autologous cartilage for craniofacial reconstruction by injection molding.Plast Reconstr Surg 112, 793-799; discussion 800-791 (2003) .
2.Beahm, EK & Walton, RL Auricular reconstruction for microtia: part I. Anatomy, embryology, and clinical evaluation.Plast Reconstr Surg 109, 2473-2482; quiz following 2482 (2002).
3. Eppley, BL & Dadvand, B. Injectable soft-tissue fillers: clinical overview. Plast Reconstr Surg 118, 98e-106e (2006).
4. Matton, G., Anseeuw, A. & De Keyser, F. The history of injectable biomaterials and the biology of collagen. Aesthetic Plast Surg 9, 133-140 (1985).
5. Nagata, S. Modification of the stages in total reconstruction of the auricle: Part I. Grafting the three-dimensional costal cartilage framework for lobule-type microtia.Plast Reconstr Surg 93, 221-230; discussion 267-228 (1994) .
6. Firmin, F., Sanger, C. &O'Toole, G. Ear reconstruction following severe complications of otoplasty. J Plast Reconstr Aesthet Surg (2008).
7. Kline, RM, Jr. & Wolfe, SA Complications associated with the harvesting of cranial bone grafts.Plast Reconstr Surg 95, 5-13; discussion 14-20 (1995).
8. Laurie, SW, Kaban, LB, Mulliken, JB & Murray, JE Donor-site morbidity after harvesting rib and iliac bone.Plast Reconstr Surg 73, 933-938 (1984).
9. Skouteris, CA & Sotereanos, GC Donor site morbidity following harvesting of autogenous rib grafts. J Oral Maxillofac Surg 47, 808-812 (1989).
10. Whitaker, LA, et al. Combined report of problems and complications in 793 craniofacial operations. Plast Reconstr Surg 64, 198-203 (1979).
11. Berry, L., Grant, ME, McClure, J. & Rooney, P. Bone-marrow-derived chondrogenesis in vitro. J Cell Sci 101 (Pt 2), 333-342 (1992).
12.Ma, HL, Hung, SC, Lin, SY, Chen, YL & Lo, WH Chondrogenesis of human mesenchymal stem cells encapsulated in alginate beads. J Biomed Mater Res A 64, 273-281 (2003).
13. Terada, S., Fuchs, JR, Yoshimoto, H., Fauza, DO & Vacanti, JP In vitro cartilage regeneration from proliferated adult elastic chondrocytes. Ann Plast Surg 55, 196-201 (2005).
14. Shieh, SJ, Terada, S. & Vacanti, JP Tissue engineering auricular reconstruction: in vitro and in vivo studies.Biomaterials 25, 1545-1557 (2004).
15. Togo, T., et al. Identification of cartilage progenitor cells in the adult ear perichondrium: utilization for cartilage reconstruction.Lab Invest 86, 445-457 (2006).
16.Dickhut, A., et al.Calculation or dedifferentiation: requirement to lock mesenchymal stem cells in a desired differentiation stage.J Cell Physiol 219, 219-226 (2009).
17. Afizah, H., Yang, Z., Hui, JH, Ouyang, HW & Lee, EH A comparison between the chondrogenic potential of human bone marrowstem cells (BMSCs) and adipose-derived stem cells (ADSCs) taken from the samedonors. Tissue Eng 13, 659-666 (2007).
18. Koga, H., et al. Comparison of mesenchymaltissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit.Cell Tissue Res 333, 207-215 (2008).
19. Sakaguchi, Y., Sekiya, I., Yagishita, K. & Muneta, T. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source.Arthritis Rheum 52,2521-2529 (2005) .
20. Langer, R. & Vacanti, JP Tissue engineering.Science 260, 920-926 (1993).
21. de Chalain, T., Phillips, JH & Hinek, A. Bioengineering of elastic cartilage with aggregated porcine and human auricular chondrocytes and hydrogels containing alginate, collagen, and kappa-elastin.J Biomed Mater Res 44, 280-288 (1999 ).
22. Jeon, YH, et al. Different effects of PLGA and chitosan scaffolds on human cartilage tissue engineering. J Craniofac Surg 18, 1249-1258 (2007).
23. Ushida, T., Furukawa, K., Toita, K. & Tateishi, T. Three-dimensional seeding of chondrocytes encapsulated in collagen gel into PLLA scaffolds. Cell Transplant 11, 489-494 (2002).
24. Quirici, N., et al. Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 30, 783-791 (2002).
25. Boiret, N., et al. Characterization of nonexpanded mesenchymal progenitor cells from normal adult human bone marrow.Exp Hematol 33, 219-225 (2005).
26. Aslan, H., et al. Osteogenic differentiation of noncultured immunoisolated bone marrow-derived CD105 + cells. Stem Cells 24, 1728-1737 (2006).
27.Nimura, A., et al. Increased proliferation of human synovial mesenchymal stem cells with autologous human serum: comparisons with bone marrow mesenchymal stem cells and with fetal bovine serum.Arthritis Rheum 58, 501-510 (2008).
28. Covas, DT, et al. Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146 + perivascular cells and fibroblasts.Exp Hematol 36, 642-654 (2008).
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本発明は、先天奇形の治療、美容整形、外傷の治療などに利用可能である。 The present invention can be used for the treatment of congenital malformations, cosmetic surgery, and the treatment of trauma.
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| WO2014148592A1 (en) | 2013-03-21 | 2014-09-25 | 公立大学法人横浜市立大学 | Method for preparing chondrocytes |
| WO2018021362A1 (en) * | 2016-07-25 | 2018-02-01 | 宇部興産株式会社 | Method to suppress dedifferentiation of cells that readily dedifferentiate, method for preparing said cells, and method for producing substance |
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| WO2014148592A1 (en) | 2013-03-21 | 2014-09-25 | 公立大学法人横浜市立大学 | Method for preparing chondrocytes |
| WO2018021362A1 (en) * | 2016-07-25 | 2018-02-01 | 宇部興産株式会社 | Method to suppress dedifferentiation of cells that readily dedifferentiate, method for preparing said cells, and method for producing substance |
| CN109477069A (en) * | 2016-07-25 | 2019-03-15 | 宇部兴产株式会社 | Inhibit the method, the modulator approach of the cell and the production method of substance of the dedifferentiation of the cell of easy dedifferentiation |
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| CN109477069B (en) * | 2016-07-25 | 2022-04-15 | 宇部兴产株式会社 | Method for inhibiting dedifferentiation of dedifferentiated cells, method for preparing the cells, and method for producing substance |
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