JPWO2015129902A1 - Fat-derived stem cell sheet having bone differentiation ability and method for producing the same - Google Patents

Fat-derived stem cell sheet having bone differentiation ability and method for producing the same Download PDF

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JPWO2015129902A1
JPWO2015129902A1 JP2016505358A JP2016505358A JPWO2015129902A1 JP WO2015129902 A1 JPWO2015129902 A1 JP WO2015129902A1 JP 2016505358 A JP2016505358 A JP 2016505358A JP 2016505358 A JP2016505358 A JP 2016505358A JP WO2015129902 A1 JPWO2015129902 A1 JP WO2015129902A1
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向 方
向 方
英樹 村上
英樹 村上
弘行 土屋
弘行 土屋
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Abstract

【課題】骨組織再生用材料となり得る細胞構築物を作製するための簡便で安価な方法、及び該方法により得られる骨形成能を有する細胞構築物を提供すること。【解決手段】細胞分化誘導剤を含まない細胞培養培地にアスコルビン酸又はその塩を添加して調製した溶液を、スキャホールドを添加していない培養容器内に加え、該溶液中で、脂肪由来幹細胞を培養することを含む、骨分化能を有する単層の脂肪由来幹細胞シートの作製方法、並びに該方法で作製された脂肪由来幹細胞シート。【選択図】なしTo provide a simple and inexpensive method for producing a cell construct that can be used as a bone tissue regeneration material, and a cell construct having bone forming ability obtained by the method. A solution prepared by adding ascorbic acid or a salt thereof to a cell culture medium not containing a cell differentiation inducer is added to a culture vessel to which no scaffold is added. A method for producing a monolayer of adipose-derived stem cell sheet having the ability to differentiate into bone, and an adipose-derived stem cell sheet produced by the method. [Selection figure] None

Description

本願発明は、幹細胞の分化誘導の技術分野に関する。具体的には、本発明は、脂肪由来幹細胞から骨分化能を有する脂肪由来幹細胞シートを作製する方法、及び該方法で得られた脂肪由来幹細胞シートに関する。
なお、本出願は、参照によりここに援用されるところの、日本国特許出願の特願2014-038913からの優先権を請求する。The present invention relates to the technical field of stem cell differentiation induction. Specifically, the present invention relates to a method for producing an adipose-derived stem cell sheet having bone differentiation ability from an adipose-derived stem cell, and an adipose-derived stem cell sheet obtained by the method.
This application claims priority from Japanese Patent Application No. 2014-038913, which is incorporated herein by reference.

（再生医療）
近年、再生医療が注目され、整形外科分野でも、骨再生を目的とした再生医療が実施されている。幹細胞を骨形成系列に分化させ、続いて骨再生に使用することは再生医療における主要な目標である。再生医療分野において、重要な３つのファクターである細胞、制御因子及び足場｛以下、スキャホールド（scaffolds）という。｝が、その地位を確立してきている（非特許文献１）。
骨髄間質細胞（bone marrow stromal cells；ＢＭＳＣｓ）が骨再生に関して注目されており（非特許文献２）、そして胚性幹細胞（ＥＳＣｓ）が、再生幹細胞の材料でもあることから、次第に注目を集めている（非特許文献３）。人工多能性幹細胞（ｉＰＳＣｓ）もまた、再生医療分野で注目を浴びており、再生幹細胞の有望な材料の１つと考えられている（非特許文献４）。(Regenerative medicine)
In recent years, regenerative medicine has been attracting attention, and regenerative medicine for the purpose of bone regeneration has also been implemented in the field of orthopedics. Differentiating stem cells into the osteogenic lineage and subsequent use in bone regeneration is a major goal in regenerative medicine. In the field of regenerative medicine, three important factors are cells, regulatory factors, and scaffolds (hereinafter referred to as scaffolds). } Has established its position (Non-Patent Document 1).
Bone marrow stromal cells (BMSCs) are attracting attention regarding bone regeneration (Non-patent Document 2), and embryonic stem cells (ESCs) are also materials for regenerative stem cells, and are attracting attention. (Non-patent Document 3). Artificial pluripotent stem cells (iPSCs) are also attracting attention in the field of regenerative medicine, and are considered as one of promising materials for regenerative stem cells (Non-patent Document 4).

（骨芽細胞）
骨形成においては骨芽細胞が重要な役割を果たしている。骨芽細胞は、骨組織において骨形成を行う細胞であり、タンパク質等を産生及び分泌して骨基質をつくる。具体的には、骨芽細胞は、生体内でコラーゲン等の基質タンパク質を分泌し、そこに基質小胞（matrix vesicle）を作る。この基質小胞の周囲にリン酸カルシウムが沈着して骨の基質が完成し、骨芽細胞は最終的にはこの基質の中で骨細胞となる。(Osteoblast)
Osteoblasts play an important role in bone formation. Osteoblasts are cells that form bone in bone tissue and produce and secrete proteins and the like to create a bone matrix. Specifically, osteoblasts secrete matrix proteins such as collagen in the living body and form matrix vesicles there. Calcium phosphate is deposited around the matrix vesicles to complete the bone matrix, and the osteoblasts eventually become bone cells in the matrix.

骨芽細胞は、間葉系幹細胞から分化するものであり、間葉系幹細胞にデキサメタゾン、β−グリセロリン酸、及びアスコルビン酸を作用させることにより分化誘導できることが開示されている（非特許文献５）。また、インビトロにおいて多能性幹細胞から分化させた間葉系幹細胞を、骨形成タンパク質（Bone Morphogenetic Protein；ＢＭＰ）−４、アスコルビン酸−２−リン酸塩、デキサメタゾン及びβ−グリセロリン酸塩を含む培養培地を使用して、ゼラチンでコートした培養プレートで培養することにより、骨芽細胞に分化誘導できたことが開示されている（特許文献１）。   It is disclosed that osteoblasts are differentiated from mesenchymal stem cells and can be induced to differentiate by allowing dexamethasone, β-glycerophosphate, and ascorbic acid to act on mesenchymal stem cells (Non-patent Document 5). . In addition, a mesenchymal stem cell differentiated from a pluripotent stem cell in vitro is cultured containing bone morphogenetic protein (BMP) -4, ascorbic acid-2-phosphate, dexamethasone, and β-glycerophosphate. It has been disclosed that differentiation can be induced in osteoblasts by culturing in a culture plate coated with gelatin using a medium (Patent Document 1).

（脂肪由来幹細胞）
間葉系幹細胞の１つとして認められている脂肪由来幹細胞（adipose-derived stem cell；以下、ＡＤＳＣｓと略称することがある）も、ＢＭＳＣｓ、ＥＳＣｓ及びｉＰＳＣｓに加えて、骨再生医療における使用が検討されている（非特許文献６）。損傷を受けた組織及び臓器の修復及び再建へのＡＤＳＣｓの利用にはいくつかの選択肢がある。(Adipose-derived stem cells)
Adipose-derived stem cells (hereinafter abbreviated as ADSCs), which are recognized as one of mesenchymal stem cells, are also being investigated for use in bone regenerative medicine in addition to BMSCs, ESCs, and iPSCs. (Non-Patent Document 6). There are several options for using ADSCs to repair and reconstruct damaged tissues and organs.

修復部位へＡＤＳＣｓを直接注入する方法（非特許文献７−１０）が知られている。また、ＡＤＳＣｓをフィブリン糊などの生物学的接着剤に混ぜて修復部位に注入する方法が知られている。しかしながら、これら方法では皮質骨表面への細胞の生着や固定が困難であった。最近、壊死した大腿骨骨頭へのＡＤＳＣｓ注入に関する良好な報告があった（非特許文献８）。それによれば、ＡＤＳＣｓを注入された大腿骨骨頭壊死例において、有望な骨形成が核磁気共鳴画像法（ＭＲＩ）による検査により観察された。しかしながら、ＡＤＳＣｓを皮質骨に固定させることは未だに困難である。   A method of directly injecting ADSCs into a repair site (Non-patent Documents 7 to 10) is known. In addition, a method is known in which ADSCs are mixed with a biological adhesive such as fibrin glue and injected into a repair site. However, it has been difficult to engraft and fix cells on the cortical bone surface by these methods. Recently, there have been good reports on ADSCs injection into the necrotic femoral head (Non-patent Document 8). According to it, promising bone formation was observed in the case of femoral head necrosis injected with ADSCs by examination by nuclear magnetic resonance imaging (MRI). However, it is still difficult to fix ADSCs to cortical bone.

また、ＡＤＳＣｓを様々な担体と共に移植する方法が知られている。心血管疾患における幹細胞治療において、天然の生物分解性マトリクス（biodegradable matrixes）で構成されたスキャホールドを使用することにより、該治療の大きな制約であった細胞生着の不足を回避することができた（非特許文献１１）。しかしながら、この様な方法によっても、細胞を移植部位に確実に固定することは困難であり、細胞が経時的に脱落する問題があった。   Also, methods for transplanting ADSCs with various carriers are known. In stem cell therapy in cardiovascular disease, by using scaffolds composed of natural biodegradable matrixes, it was possible to avoid the lack of cell engraftment, which was a major limitation of the therapy (Non-Patent Document 11). However, even with such a method, it is difficult to reliably fix the cells to the transplantation site, and there is a problem that the cells fall off over time.

一方、個々の分散状態にあるＡＤＳＣｓ（以下、分散ＡＤＳＣｓということがある）がシート状を形成してなるＡＤＳＣｓシートが、心血管分野及び形成外科分野において、組織再生に一定の成果を挙げていることが報告されている（非特許文献１２−２１）。
現在利用できるＡＤＳＣｓシートは、大まかに２種類に分けることができ、一方は、CellSeed社の製品などのような特別な担体を支持体とするＡＤＳＣｓシート（非特許文献１９−２３）であり、もう一方は、コラーゲンタンパク質担体と共に移植部位に移植するＡＤＳＣｓシートである（非特許文献１１、２４）。On the other hand, ADSCs sheets in which ADSCs in individual dispersed states (hereinafter sometimes referred to as dispersed ADSCs) form a sheet form have achieved certain results in tissue regeneration in the cardiovascular field and plastic surgery field. Has been reported (Non-Patent Documents 12-21).
Currently available ADSCs sheets can be roughly divided into two types, one of which is an ADSCs sheet (Non-patent Documents 19-23) using a special carrier such as a CellSeed product as a support. One is an ADSCs sheet transplanted to a transplant site together with a collagen protein carrier (Non-patent Documents 11 and 24).

（細胞シート）
細胞シートの製造方法は、様々な報告がある。例えば、軟骨細胞、軟骨前駆細胞、滑膜由来細胞、滑膜幹細胞、骨芽細胞、間葉系幹細胞、脂肪由来細胞、又は脂肪由来幹細胞を、支持体や担体上で培養することにより製造する方法が報告されている（特許文献２、３）。また、間葉系幹細胞、滑膜細胞、及び胚性幹細胞から選択される細胞を細胞培養支持体上で培養して、該培養した細胞を、シート状三次元構造体として作製する方法が報告されている（特許文献４）。さらに、脂肪細胞を含有する細胞シートを含む心臓疾患治療用移植材料を製造する方法であって、ａ）温度応答性高分子が被覆された細胞培養支持体上で、脂肪細胞を含有する細胞群を培養液中で培養する工程、ｂ）培養液の温度を、上限臨界溶解温度以上又は下限臨界溶解温度以下とする工程、ｃ）細胞群を、細胞培養支持体から細胞シートとして剥離する工程、さらに工程ｃ）の前にｄ）培養液にアスコルビン酸又はその誘導体を加える工程を含む方法が開示されている（特許文献５）。(Cell sheet)
There are various reports on methods for producing cell sheets. For example, a method for producing chondrocytes, cartilage precursor cells, synovial cells, synovial stem cells, osteoblasts, mesenchymal stem cells, fat-derived cells, or fat-derived stem cells by culturing them on a support or carrier. Have been reported (Patent Documents 2 and 3). Also reported is a method of culturing a cell selected from mesenchymal stem cells, synovial cells, and embryonic stem cells on a cell culture support and producing the cultured cells as a sheet-like three-dimensional structure. (Patent Document 4). Furthermore, a method for producing a transplant material for treating heart disease comprising a cell sheet containing a fat cell, wherein a) a group of cells containing a fat cell on a cell culture support coated with a temperature-responsive polymer B) a step of bringing the temperature of the culture solution above the upper critical solution temperature or lower than the lower critical solution temperature, c) a step of peeling the cell group from the cell culture support as a cell sheet, Furthermore, a method including a step of adding ascorbic acid or a derivative thereof to the culture broth before step c) is disclosed (Patent Document 5).

しかし、ＡＤＳＣｓシートの整形外科での使用を試みた例は報告されていない。本願発明者らは、骨塊を液体窒素中で凍結後に骨マトリクスを維持し、その後、それを元の位置に戻して固定する液体窒素療法を開発している（非特許文献２５−２７）。整形外科分野でのＡＤＳＣｓの応用は、分散ＡＤＳＣｓと他の物質、例えば標準生理食塩水、フィブリンゲル及びコラーゲンゲルなどとの混合物を海綿質骨に注入する（非特許文献８、２８）といった方法により行われているのみである。
また、ＡＤＳＣｓとβ型リン酸三カルシウム（β−ＴＣＰ）ベースの骨セメントスキャホールドとの併用が骨欠損に対して見込みがあるとの報告がある（非特許文献２９、３０）。
しかしながら、ＡＤＳＣｓは、整形外科分野においては、移植箇所で固定するためにフィブリン糊様の物質を構成成分とする混合物として使用されるため、そのような混合物に含まれるＡＤＳＣｓの濃度には制限がある。However, there have been no reports of attempts to use ADSCs sheets in orthopedics. The inventors of the present application have developed liquid nitrogen therapy that maintains a bone matrix after freezing bone mass in liquid nitrogen and then fixes it back to its original position (Non-patent Documents 25-27). The application of ADSCs in the orthopedic field is based on a method in which a mixture of dispersed ADSCs and other substances such as standard saline, fibrin gel, and collagen gel is injected into cancellous bone (Non-patent Documents 8 and 28). It is only done.
In addition, it has been reported that the combined use of ADSCs and β-tricalcium phosphate (β-TCP) based bone cement scaffolds is promising for bone defects (Non-Patent Documents 29 and 30).
However, since ADSCs are used as a mixture containing a fibrin glue-like substance as a component in the orthopedic field in order to fix at an implantation site, the concentration of ADSCs contained in such a mixture is limited. .

国際公開第２００４／１０６５０２号パンフレットInternational Publication No. 2004/106502 Pamphlet 特許第０４９２１３５３号公報Japanese Patent No. 04921353 特許第０４６２０１１０号公報Japanese Patent No. 04620110 特許第０４９４３８４４号公報Japanese Patent No. 04943844 国際公開第２０１１／０６７９８３号パンフレットInternational Publication No. 2011/067983 Pamphlet

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脂肪由来幹細胞（ＡＤＳＣｓ）がインビトロ及びインビボのいずれにおいても骨芽細胞に分化誘導される可能性を有することが報告されている。しかしながら、分散ＡＤＳＣｓを骨再生及び骨再建に使用することは今までのところ困難であった。   It has been reported that adipose-derived stem cells (ADSCs) have the potential to be induced to differentiate into osteoblasts both in vitro and in vivo. However, it has so far been difficult to use dispersed ADSCs for bone regeneration and reconstruction.

本発明の課題は、ＡＤＳＣｓを利用して、骨組織再生用材料となり得る細胞構築物を作製するための簡便で安価な方法、及び該方法により得られる骨形成能を有する細胞構築物を提供することである。   An object of the present invention is to provide a simple and inexpensive method for producing a cell construct that can be a bone tissue regeneration material using ADSCs, and a cell construct having bone forming ability obtained by the method. is there.

本発明者らは、上記目的を達成すべく、鋭意研究を行った。そして、細胞培養培地にアスコルビン酸−２−リン酸のみを添加することにより調製した作製培地（fabricating medium）で、ＡＤＳＣｓを培養することにより、ＡＤＳＣｓシートが作製されること、並びに、このようにして作製されたＡＤＳＣｓシートは、骨分化誘導を実施したときに、従来の細胞培養培地で培養したＡＤＳＣｓと比較して、短期間で有効に骨誘導されることを見出した。
また、このような方法で作製されたＡＤＳＣｓシートはその力学的強度が高く、骨損傷部位に移植したときに、骨表面への生着が良く、インビボでも有効に骨分化が誘導されると考えることができる。
以上により、本発明は、これら知見により達成したものである。The inventors of the present invention have intensively studied to achieve the above object. An ADSCs sheet is produced by culturing ADSCs in a production medium prepared by adding only ascorbic acid-2-phosphate to the cell culture medium. It was found that the produced ADSCs sheet was effectively induced in a short period of time when bone differentiation was induced, compared to ADSCs cultured in a conventional cell culture medium.
In addition, the ADSCs sheet prepared by such a method has high mechanical strength, and when transplanted to a bone damage site, the ADSCs sheet has good engraftment on the bone surface and is considered to induce bone differentiation effectively even in vivo. be able to.
As described above, the present invention has been achieved by these findings.

即ち、本発明は以下に関する。
１．細胞分化誘導剤を含まない細胞培養培地にアスコルビン酸又はその塩を添加して調製した溶液を、スキャホールドを添加していない培養容器内に加え、該溶液中で、脂肪由来幹細胞を培養することを含む、骨分化能を有する脂肪由来幹細胞シートの作製方法。
２．前記アスコルビン酸又はその塩が、５０μＭ〜５００μＭの濃度になるように細胞培養培地に添加されることを特徴とする、前項１に記載の作製方法。
３．前記脂肪由来幹細胞を培養することが、脂肪由来幹細胞を３日〜１５日間培養することである、前項１又は２に記載の作製方法。
４．前記脂肪由来幹細胞シートが単層の脂肪由来幹細胞シートである、前項１〜３のいずれか１に記載の作製方法。
５．前項１〜４のいずれか１に記載の作製方法により製造された、骨分化能を有する脂肪幹細胞シート。
６．前項５に記載の脂肪幹細胞シートを、骨分化誘導剤を含む細胞培養培地中で培養することを含む、骨芽細胞を含む細胞シートの作製方法。
７．前項５に記載の骨分化能を有する脂肪幹細胞シート又は前項６に記載の作製方法により製造された骨芽細胞を含む細胞シートを含む骨組織再生用材料。
８．前記骨組織再生用材料は、スキャホールドを含まないことを特徴とする、前項７に記載の骨組織再生用材料。That is, the present invention relates to the following.
1. A solution prepared by adding ascorbic acid or a salt thereof to a cell culture medium not containing a cell differentiation inducer is added to a culture vessel to which no scaffold is added, and adipose-derived stem cells are cultured in the solution. A method for producing an adipose-derived stem cell sheet having bone differentiation ability.
2. 2. The production method according to item 1, wherein the ascorbic acid or a salt thereof is added to a cell culture medium so as to have a concentration of 50 μM to 500 μM.
3. The production method according to item 1 or 2, wherein culturing the adipose-derived stem cells is culturing the adipose-derived stem cells for 3 to 15 days.
4). 4. The production method according to any one of items 1 to 3, wherein the fat-derived stem cell sheet is a monolayer fat-derived stem cell sheet.
5. An adipose stem cell sheet having the ability to differentiate into bone, produced by the production method according to any one of items 1 to 4.
6). 6. A method for producing a cell sheet containing osteoblasts, comprising culturing the adipose stem cell sheet according to item 5 in a cell culture medium containing a bone differentiation-inducing agent.
7). A material for regenerating bone tissue comprising the adipose stem cell sheet having the ability to differentiate into bone according to item 5 or the cell sheet containing osteoblasts produced by the production method according to item 6 above.
8). 8. The bone tissue regeneration material according to item 7 above, wherein the bone tissue regeneration material does not include a scaffold.

本発明によれば、通常の細胞培養培地にアスコルビン酸又はその塩のみを添加して調製した溶液中で、細胞培養支持体を用いずに、ＡＤＳＣｓを培養することを含む、骨分化能を有するＡＤＳＣｓシートの作製方法、並びに該作製方法により得られる、骨分化能を有するＡＤＳＣｓシートを提供できる。   According to the present invention, it has bone differentiation ability including culturing ADSCs in a solution prepared by adding only ascorbic acid or a salt thereof to a normal cell culture medium without using a cell culture support. An ADSCs sheet production method and an ADSCs sheet obtained by the production method and having bone differentiation ability can be provided.

本発明によれば、骨再生及び骨再建のための骨組織再生用材料として使用することができるＡＤＳＣｓシートを提供することができる。
本発明のＡＤＳＣｓシートは、分散ＡＤＳＣｓと比較して、骨形成能が高く、迅速に骨形成が可能である。
また、分散ＡＤＳＣｓは骨組織再生部位におけるその固定及び増殖のためにフィブリン糊等の固定用材料やスキャホールドと共に使用される必要があったが、本発明のＡＤＳＣｓシートは、スキャホールドと共に使用してもよいが、その力学的強度が高いため、それら自身のみで移植部分に接着することができる。このように、ＡＤＳＣｓシートは骨分化能を有する細胞を含み、またそれ自身単独で修復部位に固定され得るため、骨組織再生用材料として、分散ＡＤＳＣｓと比較してより有用である。ADVANTAGE OF THE INVENTION According to this invention, the ADSCs sheet | seat which can be used as a bone tissue reproduction | regeneration material for bone regeneration and bone reconstruction can be provided.
The ADSCs sheet of the present invention has higher bone forming ability and can rapidly form bone as compared with dispersed ADSCs.
In addition, the dispersed ADSCs need to be used together with a fixing material such as fibrin glue or a scaffold for fixation and proliferation at the bone tissue regeneration site, but the ADSCs sheet of the present invention is used together with the scaffold. However, because of its high mechanical strength, it can be adhered to the transplanted part by itself. Thus, the ADSCs sheet contains cells having the ability to differentiate into bone, and can be fixed to a repair site by itself. Therefore, the ADSCs sheet is more useful as a bone tissue regeneration material compared to dispersed ADSCs.

また、本発明に係る方法は、アスコルビン酸又はその塩の他に他の物質を使用せずに骨組織再生用材料となるＡＤＳＣｓシートを作製できるため、簡便かつ安価に実施できる。   In addition, the method according to the present invention can be carried out simply and inexpensively because an ADSCs sheet as a bone tissue regeneration material can be produced without using other substances in addition to ascorbic acid or a salt thereof.

ＡＤＳＣｓシートの組織学的形状と細胞密度を、顕微鏡下で、オーバーコンフルエント状態のＡＤＳＣｓと比較した結果を説明する図である。両者の組織学的形状と細胞密度には明らかな差異は認められなかった。パネルＡ、Ｂ及びＣは、ＡＤＳＣｓシートをそれぞれ４倍、１０倍及び２０倍の倍率で観察した視野を示す。パネルＤは、オーバーコンフルエント状態のＡＤＳＣｓを２０倍の倍率で観察した視野を示す。It is a figure explaining the result of having compared the histological shape and cell density of an ADSCs sheet with ADSCs of an overconfluent state under a microscope. There was no obvious difference between their histological shape and cell density. Panels A, B and C show the field of view of the ADSCs sheet observed at 4 ×, 10 × and 20 × magnifications, respectively. Panel D shows the field of view of overconfluent ADSCs at 20x magnification. 骨誘導培地で培養したＡＤＳＣｓが骨芽細胞に分化誘導したことを説明する図である。骨芽細胞の検出はＡＬＰ染色（Alkaline Phosphatase Staining；ALP staining）及びアリザリンレッド染色（Alizarin red staining）により行った。ＡＤＳＣｓは、骨誘導後２週間目からＡＬＰ染色陽性を示し、３週間目からアリザリンレッド染色陽性を示した。図中、「ｉｃ」は骨誘導培地中のＡＤＳＣｓ群を示し、「ｎｃ」は通常倍地中のＡＤＳＣｓ群を示す。It is a figure explaining that ADSCs culture | cultivated with the osteoinduction culture medium induced differentiation to the osteoblast. Osteoblasts were detected by ALP staining (Alkaline Phosphatase Staining; ALP staining) and alizarin red staining. ADSCs showed positive ALP staining from 2 weeks after bone induction and positive alizarin red staining from 3 weeks. In the figure, “ic” indicates the ADSCs group in the osteoinduction medium, and “nc” indicates the ADSCs group in the normal medium. 骨誘導培地で培養したＡＤＳＣｓシートが骨芽細胞に分化誘導したことを説明する図である。骨芽細胞の検出はＡＬＰ染色（ALP staining）及びアリザリンレッド染色（Alizarin red staining）により行った。ＡＤＳＣｓシートは、骨誘導後５日目からＡＬＰ染色陽性を示し、７日目からアリザリンレッド染色陽性を示した。図中、「ｉｓ」は骨誘導培地中のＡＤＳＣｓシート群を示し、「ｎｓ」は通常倍地中のＡＤＳＣｓシート群を示す。It is a figure explaining that the ADSCs sheet | seat culture | cultivated with the osteoinduction culture medium induced differentiation to the osteoblast. Osteoblasts were detected by ALP staining (ALP staining) and alizarin red staining. The ADSCs sheet was positive for ALP staining from day 5 after bone induction and positive for alizarin red staining from day 7. In the figure, “is” indicates the ADSCs sheet group in the osteoinduction medium, and “ns” indicates the ADSCs sheet group in the normal medium. 骨誘導培地で培養したＡＤＳＣｓ及びＡＤＳＣｓシートそれぞれのＡＬＰ活性（ALP activity）を示す図である。ＡＬＰ活性は、１×１０５細胞当たりの活性で示した。パネルＡは、経日的なＡＬＰ活性曲線を示す。パネルＢは、培養３日目、５日目、７日目、１０日目のＡＬＰ活性を、通常培地で培養した対照群と比較した結果を示す。図中、「ｉｓ」は骨誘導培地中のＡＤＳＣｓシート群を示し、「ｎｓ」は通常培地中のＡＤＳＣｓシート群を示し、「ｉｃ」は骨誘導培地中のＡＤＳＣｓを示し、そして「ｎｃ」は通常培地中のＡＤＳＣｓを示す。 パネルＡでは、◆、■、▲及び×はそれぞれｉｓ、ｎｓ、ｉｃ及びｎｃを示す。パネルＢでは、データは左側から順にｉｓ、ｎｓ、ｉｃ及びｎｃである。It is a figure which shows ALP activity (ALP activity) of each of ADSCs and ADSCs sheet | seat culture | cultivated with the osteoinduction culture medium. ALP activity was expressed as activity per 1 × 10 5 cells. Panel A shows a daily ALP activity curve. Panel B shows the results of comparing the ALP activity on day 3, day 5, day 7 and day 10 of the culture with a control group cultured in a normal medium. In the figure, “is” indicates the ADSCs sheet group in the osteoinduction medium, “ns” indicates the ADSCs sheet group in the normal medium, “ic” indicates the ADSCs in the osteoinduction medium, and “nc” Shown are ADSCs in normal media. In panel A, ◆, ■, ▲, and x indicate is, ns, ic, and nc, respectively. In panel B, the data are is, ns, ic and nc in order from the left. ＡＤＳＣｓシートのＡＬＰ活性は４０５ ｎｍ波長の吸光度により測定した。図のグラフは、異なるアスコルビン酸濃度の培地で作製されたＡＤＳＣｓシートの吸光度を、アスコルビン酸濃度５０ μＭで作製した場合と比較している。各日のアスコルビン酸濃度５０ μＭで作製したＡＤＳＣｓシートの吸光度を１とした。０ｄａｙはＡＤＳＣｓがオーバーコンフルエントに達した状態を意味し、ＡＤＳＣｓシート培地はその時点から測定に使用した。なお、各日数のグラフの左はアスコルビン酸濃度５０ μＭ、中はアスコルビン酸濃度１５０ μＭ及び右はアスコルビン酸濃度４５０ μＭを示す。The ALP activity of the ADSCs sheet was measured by absorbance at a wavelength of 405 nm. The graph of the figure compares the absorbance of ADSCs sheets prepared in media with different ascorbic acid concentrations with those prepared at ascorbic acid concentrations of 50 μM. The absorbance of the ADSCs sheet prepared at an ascorbic acid concentration of 50 μM on each day was 1. 0 day means that the ADSCs reached overconfluence, and the ADSCs sheet medium was used for the measurement from that point. In addition, the left of the graph of each day shows ascorbic acid concentration 50 μM, inside shows ascorbic acid concentration 150 μM, and right shows ascorbic acid concentration 450 μM.

（本発明の概要）
本発明は、骨分化能を有するＡＤＳＣｓシート（より詳細には、単層のＡＤＳＣｓシート）の作製方法、及び該作製方法で作製されたＡＤＳＣｓシートに関する。
本方法は、細胞分化誘導剤を含まない細胞培養培地にアスコルビン酸又はその塩を添加して調製した溶液を、細胞培養支持体を添加していない培養容器内に加え、該溶液中で、脂肪由来幹細胞を培養することを含む。
特に、ＡＤＳＣｓシートは骨形成用に使用することが好ましい。(Outline of the present invention)
The present invention relates to a method for producing an ADSCs sheet having bone differentiation ability (more specifically, a single-layer ADSCs sheet), and an ADSCs sheet produced by the production method.
In this method, a solution prepared by adding ascorbic acid or a salt thereof to a cell culture medium not containing a cell differentiation inducer is added to a culture vessel to which a cell culture support is not added, Culturing the derived stem cells.
In particular, the ADSCs sheet is preferably used for bone formation.

本発明に係るＡＤＳＣｓシートの作製方法は、骨再生及び骨再建のための骨組織再生用材料として使用することができるＡＤＳＣｓシートを、簡便、迅速及び安価に作製できるという利点を有する。   The method for producing an ADSCs sheet according to the present invention has an advantage that an ADSCs sheet that can be used as a bone tissue regeneration material for bone regeneration and bone reconstruction can be produced simply, quickly, and inexpensively.

また、本発明に係る作製方法により作成されたＡＤＳＣｓシートは、骨分化能を有する細胞を含み、また、その力学的強度が高いため、それ自身を単独で修復部位に移植されたときにその部位に固定され得るという利点を有する。   In addition, the ADSCs sheet prepared by the preparation method according to the present invention includes cells having bone differentiation ability, and since the mechanical strength thereof is high, the site when the cell itself is transplanted to a repair site alone. It has the advantage that it can be fixed to.

本発明は、さらに、本発明の作製方法により作成されたＡＤＳＣｓシートを、骨分化誘導剤を含む細胞培養培地中で培養することを含む、骨芽細胞を含む細胞シートの作製方法を提供する。   The present invention further provides a method for producing a cell sheet containing osteoblasts, comprising culturing an ADSCs sheet produced by the production method of the present invention in a cell culture medium containing an osteoinduction agent.

（幹細胞）
「幹細胞（stem cell）」とは、複数系統の細胞に分化できる能力（多分化能）と、細胞分裂を経ても多分化能を維持できる能力（自己複製能）を併せ持つ細胞をいう。幹細胞は、発生の過程や組織・器官の維持において細胞を供給する役割を担っている。幹細胞には、全ての系統の細胞に分化することのできる胚性幹細胞、通常は分化系統が限定されている成体幹細胞（組織幹細胞、体性幹細胞）、胚体外組織を除く全ての系統に分化する多能性を有し、人工的に作製された人工多能性幹細胞等が知られている。(Stem cell)
A “stem cell” refers to a cell that has both the ability to differentiate into cells of multiple lineages (multipotency) and the ability to maintain pluripotency even after cell division (self-renewal ability). Stem cells play a role in supplying cells in the process of development and maintenance of tissues and organs. Stem cells can be differentiated into embryonic stem cells that can differentiate into cells of all lineages, usually adult stem cells with limited differentiation lineages (tissue stem cells, somatic stem cells), and all lineages except extraembryonic tissues Artificially induced pluripotent stem cells having pluripotency and the like are known.

（脂肪由来幹細胞）
「脂肪由来幹細胞（adipose-derived stem cell；ＡＤＳＣｓ）」は、脂肪組織に存在する幹細胞である。主に間葉系幹細胞で構成され、骨髄由来の幹細胞と同様に、骨芽細胞、軟骨細胞、心筋細胞、脂肪細胞、肝細胞、血管内皮細胞及びインスリン分泌細胞等の多種類の細胞に分化することができる多能性を有する。脂肪組織は採取が容易であり、また多量の幹細胞を含むため、ＡＤＳＣｓは容易かつ大量に調製することが可能である。(Adipose-derived stem cells)
“Adipose-derived stem cells (ADSCs)” are stem cells present in adipose tissue. It is mainly composed of mesenchymal stem cells and differentiates into many types of cells such as osteoblasts, chondrocytes, cardiomyocytes, adipocytes, hepatocytes, vascular endothelial cells, and insulin-secreting cells, similar to bone marrow-derived stem cells. Have pluripotency. Since adipose tissue is easy to collect and contains a large amount of stem cells, ADSCs can be prepared easily and in large quantities.

（脂肪由来幹細胞の分離及び精製）
脂肪組織からのＡＤＳＣｓの分離及び精製は、従来報告された公知の方法を参考にして実施できる。例えば、文献Arterioscler Thromb Vasc Biol. 2009;29:1723-1729に記載の方法を用いることができる。あるいは、市販のヒトＡＤＳＣｓ（例：Invitrogen社製品）を使用することもできる。(Separation and purification of adipose-derived stem cells)
Separation and purification of ADSCs from adipose tissue can be performed with reference to known methods reported in the past. For example, the method described in the document Arterioscler Thromb Vasc Biol. 2009; 29: 1723-1729 can be used. Alternatively, commercially available human ADSCs (eg, Invitrogen product) can also be used.

（脂肪由来幹細胞シート）
「脂肪由来幹細胞シート（ＡＤＳＣｓシート）」は、個々別々の分散状態にあるＡＤＳＣｓがシート状を形成してなる細胞構築物をいう。
「単層の脂肪由来幹細胞シート」とは、ＡＤＳＣｓが一層のシート状態を形成してなり、重層構造や三次元構造を形成していない細胞構築物をいう。(Adipose-derived stem cell sheet)
“Adipose-derived stem cell sheet (ADSCs sheet)” refers to a cell construct in which ADSCs in separate dispersion form a sheet.
“Single-layered adipose-derived stem cell sheet” refers to a cell construct in which ADSCs are formed in a single sheet state and do not form a multi-layer structure or a three-dimensional structure.

本明細書において、個々別々の分散状態にあるＡＤＳＣｓを単にＡＤＳＣｓ又は分散ＡＤＳＣｓといい、シート状を形成してなるＡＤＳＣｓをＡＤＳＣｓシートという。   In the present specification, ADSCs that are in individual dispersed states are simply referred to as ADSCs or distributed ADSCs, and ADSCs formed in a sheet form are referred to as ADSCs sheets.

（脂肪由来幹細胞の由来）
ＡＤＳＣｓ及びＡＤＳＣｓシートは、哺乳動物の脂肪由来のものであればいずれでもよく、哺乳動物として、ヒト、サル、ブタ、ブタ、ウマ、ウシ、ウサギ、ヒツジ、ヤギ、ネコ、イヌ、モルモット等を例示できる。
好ましくは、ヒトの脂肪由来のＡＤＳＣｓ及びＡＤＳＣｓシートである。
さらに、ＡＤＳＣｓ及びＡＤＳＣｓシートは、骨組織再生材料として使用される場合、同種ＡＤＳＣｓ及び同種ＡＤＳＣｓシートであることが好ましく、自家ＡＤＳＣｓ及び自家ＡＤＳＣｓシートであることがさらに好ましい。
同種ＡＤＳＣｓ及び同種ＡＤＳＣｓシートとは、同じ動物種に由来するＡＤＳＣｓ及びＡＤＳＣｓシートを意味する。
自家ＡＤＳＣｓ及び自家ＡＤＳＣｓシートとは、骨組織再生を受ける対象自身に由来するＡＤＳＣｓ及びＡＤＳＣｓシートを意味する。(Derived from adipose-derived stem cells)
The ADSCs and ADSCs sheets may be any one derived from mammalian fat, and examples of mammals include humans, monkeys, pigs, pigs, horses, cows, rabbits, sheep, goats, cats, dogs, guinea pigs, etc. it can.
Preferred are human fat-derived ADSCs and ADSCs sheets.
Further, when the ADSCs and ADSCs sheets are used as a bone tissue regeneration material, the same kind of ADSCs and the same kind of ADSCs sheet are preferable, and it is more preferable that they are self ADSCs and self ADSCs sheets.
Homogeneous ADSCs and homologous ADSCs sheets mean ADSCs and ADSCs sheets derived from the same animal species.
In-house ADSCs and in-house ADSCs sheets mean ADSCs and ADSCs sheets derived from the subject who is subjected to bone tissue regeneration.

（骨分化能）
「骨分化能」とは、骨芽細胞、骨細胞又はそれらの前駆細胞に分化する能力をいう。すなわち、骨分化誘導条件下で、骨芽細胞、骨細胞又はそれらの前駆細胞に分化する能力をいう。骨分化誘導条件は、インビボ及びインビトロのいずれの条件下であってもよい。インビトロの骨分化誘導として、デキサメタゾン、β−グリセロリン酸、及びアスコルビン酸を含む培地での培養（非特許文献５）や、ＢＭＰ−４、アスコルビン酸−２−リン酸塩、デキサメタゾン及びβ−グリセロリン酸塩を含む培養培地での培養を例示できる。(Bone differentiation ability)
“Bone differentiation ability” refers to the ability to differentiate into osteoblasts, bone cells or their precursor cells. That is, it refers to the ability to differentiate into osteoblasts, bone cells or their progenitor cells under conditions that induce bone differentiation. The condition for inducing bone differentiation may be any of in vivo and in vitro conditions. For in vitro bone differentiation induction, culture in a medium containing dexamethasone, β-glycerophosphate and ascorbic acid (Non-patent Document 5), BMP-4, ascorbic acid-2-phosphate, dexamethasone and β-glycerophosphate Examples thereof include culture in a culture medium containing a salt.

（脂肪由来幹細胞シートの作製方法の特徴）
本発明の脂肪由来幹細胞シートの作製方法では、細胞培養支持体を添加していない培養容器内に、細胞分化誘導剤を含まない細胞培養培地にアスコルビン酸又はその塩を添加して調製した溶液を加え、該溶液中で、脂肪由来幹細胞を培養することを特徴とする。(Characteristics of production method of adipose-derived stem cell sheet)
In the method for producing an adipose-derived stem cell sheet of the present invention, a solution prepared by adding ascorbic acid or a salt thereof to a cell culture medium not containing a cell differentiation inducer in a culture vessel to which no cell culture support is added. In addition, fat-derived stem cells are cultured in the solution.

細胞の培養に使用する容器は、細胞培養用に一般的に使用されているものであれば特に限定されず、シャーレやフラスコなどの細胞培養容器を例示できる。接着性の細胞であれば、このような細胞培養容器の壁面に接着して増殖し、浮遊性の細胞であれば、培養溶液中に遊離の状態で増殖する。このような細胞培養容器を基体と称することがある。   The container used for cell culture is not particularly limited as long as it is generally used for cell culture, and examples thereof include cell culture containers such as petri dishes and flasks. If it is an adhesive cell, it grows by adhering to the wall of such a cell culture container, and if it is a floating cell, it grows in a free state in the culture solution. Such a cell culture container may be referred to as a substrate.

（細胞培養支持体）
「細胞培養支持体」とは、細胞の付着及び増殖、あるいは細胞による三次元構造の構築を目的として基体に被覆又は積層されたコーティング材料で形成された構造体いう。細胞培養支持体として、高分子が架橋した三次元網目構造体であるハイドロゲルやコラーゲンマトリックス（非特許文献３１）等を好ましく例示できる。本明細書において、「細胞培養支持体」と言う場合は、基体を含まない。(Cell culture support)
“Cell culture support” refers to a structure formed of a coating material that is coated or laminated on a substrate for the purpose of cell attachment and proliferation, or construction of a three-dimensional structure of cells. Preferred examples of the cell culture support include hydrogel and collagen matrix (Non-patent Document 31), which are three-dimensional network structures in which polymers are crosslinked. In the present specification, the term “cell culture support” does not include a substrate.

細胞培養支持体を添加していない培養容器として、例えば通常市販されているシャーレや細胞培養フラスコ等を例示できる。   Examples of the culture container to which the cell culture support is not added include, for example, a commercially available petri dish or a cell culture flask.

（アスコルビン酸又はその塩）
本発明に係る方法で使用するアスコルビン酸は、Ｌ−アスコルビン酸又はその誘導体、例えばＬ−アスコルビン酸−２−グルコシドやＬ−アスコルビン酸−２−リン酸等であり、好ましくはＬ−アスコルビン酸−２−リン酸である。
アスコルビン酸の塩とは、ナトリウム塩又はカルシウム塩等の薬理学的に許容される塩であればいずれの塩であってもよい。
アスコルビン酸−２−リン酸は、間葉系細胞のコラーゲンタンパク質分泌を増加させるが、該細胞に他の影響は何ら与えないことが報告されている（非特許文献３２）。
細胞培養培地に添加されるアスコルビン酸又はその塩は、最終濃度が１０μＭ〜８００μＭ、好ましくは５０μＭ〜５００μＭ、より好ましくは１００μＭ〜２００μＭ、最も好ましくは約１５０μＭになるように細胞培養培地に添加する。(Ascorbic acid or its salt)
Ascorbic acid used in the method according to the present invention is L-ascorbic acid or a derivative thereof such as L-ascorbic acid-2-glucoside, L-ascorbic acid-2-phosphate, etc., preferably L-ascorbic acid- 2-phosphoric acid.
The salt of ascorbic acid may be any salt as long as it is a pharmacologically acceptable salt such as sodium salt or calcium salt.
Ascorbic acid-2-phosphate has been reported to increase collagen protein secretion in mesenchymal cells, but has no other effect on the cells (Non-patent Document 32).
Ascorbic acid or a salt thereof added to the cell culture medium is added to the cell culture medium so that the final concentration is 10 μM to 800 μM, preferably 50 μM to 500 μM, more preferably 100 μM to 200 μM, and most preferably about 150 μM.

（細胞培養培地）
細胞培養培地は、ＡＤＳＣｓの培養に一般的に使用されている培養培地を使用することができる。例えば、ダルベッコ改変イーグル培地（Dulbecco's modified Eagle's medium；ＤＭＥＭ）を好ましく例示できるが、特に限定されない。(Cell culture medium)
As the cell culture medium, a culture medium generally used for culturing ADSCs can be used. For example, Dulbecco's modified Eagle's medium (DMEM) can be preferably exemplified, but is not particularly limited.

（脂肪由来幹細胞の培養条件）
ＡＤＳＣｓの培養条件は一般的に知られている培養条件を採用することができる。
ＡＤＳＣｓシートの作製のためのＡＤＳＣｓ培養日数は、３日〜１５日、好ましくは４日〜１３日、より好ましくは５日〜１０日、さらに好ましくは７日〜１０日、最も好ましくは７日間である。(Culture conditions for adipose-derived stem cells)
As culture conditions for ADSCs, generally known culture conditions can be employed.
The number of days of ADSCs culture for preparing the ADSCs sheet is 3 to 15 days, preferably 4 to 13 days, more preferably 5 to 10 days, still more preferably 7 to 10 days, and most preferably 7 days. is there.

（骨芽細胞を含む細胞シート）
本発明に係る作製方法で作製された脂肪幹細胞シートを、骨分化誘導剤を含む細胞培養培地中で培養することにより、骨芽細胞を含む細胞シートを作製し提供することができる。(Cell sheet containing osteoblasts)
A cell sheet containing osteoblasts can be produced and provided by culturing the adipose stem cell sheet produced by the production method according to the present invention in a cell culture medium containing an osteodifferentiation inducer.

（骨細胞分化誘導剤）
骨細胞分化誘導剤とは、幹細胞や骨系列前駆細胞を、骨芽細胞又は骨細胞に分化させ得る薬剤をいう。骨細胞分化誘導剤として、公知の骨細胞分化誘導剤をいずれも使用することができるが、デキサメタゾン、β−グリセロリン酸、及びアスコルビン酸を含む混合物、並びにＢＭＰ−４、アスコルビン酸−２−リン酸塩、デキサメタゾン及びβ−グリセロリン酸塩を含む混合物を好ましく例示できる。インビトロにおける骨細胞分化誘導は、骨細胞分化誘導剤を加えた培養培地中で、幹細胞や骨系列前駆細胞を、培養することにより実施できる。骨細胞分化誘導剤を加える培養培地は、一般的に使用されている培地であればいずれも使用できるが、α−最小必須培地（alpha-Minimum Essential Media；α−ＭＥＭ）を好ましく例示できる。
培養条件は、骨分化誘導剤を含む細胞培養培地中で、本発明に係る作製方法で作製された脂肪幹細胞シートを培養し、該シート中での骨芽細胞の分化誘導を検出することにより適宜変更して設定することができる。
骨芽細胞の分化誘導の検出は、公知方法、例えばＡＬＰ染色（ALP staining）やアリザリンレッド染色、あるいはＡＬＰ活性の測定により実施することができる。(Osteocyte differentiation inducer)
The bone cell differentiation-inducing agent refers to a drug that can differentiate stem cells and bone lineage progenitor cells into osteoblasts or bone cells. As a bone cell differentiation inducer, any known bone cell differentiation inducer can be used, but a mixture containing dexamethasone, β-glycerophosphate, and ascorbic acid, and BMP-4, ascorbic acid-2-phosphate Preferred examples include a mixture containing a salt, dexamethasone and β-glycerophosphate. Bone cell differentiation induction in vitro can be performed by culturing stem cells and bone lineage progenitor cells in a culture medium to which an osteoblast differentiation inducer is added. Any culture medium to which a bone cell differentiation inducer is added can be used as long as it is a commonly used medium, and an α-minimum essential medium (α-MEM) can be preferably exemplified.
The culture conditions are appropriately determined by culturing the adipose stem cell sheet produced by the production method according to the present invention in a cell culture medium containing an osteoinduction agent and detecting differentiation induction of osteoblasts in the sheet. Can be changed and set.
Detection of osteoblast differentiation induction can be performed by a known method such as ALP staining (ALP staining), alizarin red staining, or measurement of ALP activity.

（骨組織再生用材料）
骨組織再生用材料とは、骨再生及び骨再建のために使用する材料をいう。
本発明の骨組織再生用材料は、本発明の骨分化能を有する脂肪幹細胞シート、又は、本発明の骨芽細胞を含む細胞シート（又は、本発明に係る作製方法により製造された骨芽細胞を含む細胞シート）を含む。特に、本発明の骨組織再生用材料では、骨芽細胞を含む細胞シートが力学的強度を有しかつ自身単独で骨修復部位に固定されるので、スキャホールドを必要としないことが特徴である。(Bone tissue regeneration material)
The bone tissue regeneration material refers to a material used for bone regeneration and bone reconstruction.
The bone tissue regeneration material of the present invention is an adipose stem cell sheet having the osteogenic potential of the present invention, or a cell sheet containing the osteoblast of the present invention (or an osteoblast produced by the production method according to the present invention). Cell sheet). In particular, the bone tissue regeneration material of the present invention is characterized in that a cell sheet containing osteoblasts has mechanical strength and is fixed to a bone repair site by itself, so that no scaffold is required. .

以下、実施例を示して本発明をより具体的に説明するが、本発明は以下に示す実施例によって何ら限定されるものではない。   EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

ヒト脂肪由来幹細胞（ｈＡＤＳＣｓ）を使用し、これら細胞が骨芽細胞へ分化誘導される方法を検討した。   Using human adipose-derived stem cells (hADSCs), a method for inducing differentiation of these cells into osteoblasts was examined.

（材料）
ヒト脂肪由来幹細胞は、Life Technologies社より購入した。ＡＤＳＣｓの細胞表面タンパク質の特徴は、フローサイトメトリーにより解析しており、ＣＤ２９、ＣＤ４４、ＣＤ７３、ＣＤ９０、ＣＤ１０５及びＣＤ１６６陽性であり、ＣＤ１４、ＣＤ３１、ＣＤ４５及びＬｉｎ１陰性である。(material)
Human adipose-derived stem cells were purchased from Life Technologies. The characteristics of the cell surface proteins of ADSCs were analyzed by flow cytometry and are CD29, CD44, CD73, CD90, CD105 and CD166 positive and CD14, CD31, CD45 and Lin1 negative.

凍結保存されていたｈＡＤＳＣｓを融解した後、１０％ 牛胎仔血清（ＦＢＳ）（NICHIREI BIOSCIENCES社）及び１％ ペニシリン−ストレプトマイシン溶液（Ｐ／Ｓ）(Wako Pure Chemical Industries社）を加えたＤＭＥＭ（Wako Pure Chemical Industries, Ltd社）にけん濁し、３７℃で１日間インキュベーションした。   After thawing the hADSCs that had been cryopreserved, DMEM (Wako Pure) supplemented with 10% fetal bovine serum (FBS) (NICHIREI BIOSCIENCES) and 1% penicillin-streptomycin solution (P / S) (Wako Pure Chemical Industries) Chemical Industries, Ltd.) and incubated at 37 ° C. for 1 day.

前記１日間のインキュベーション後、活性化したｈＡＤＳＣｓの形態が円形から紡錘形に変化することが、光学顕微鏡により観察できた。これらの形態の変化は、細胞からのコラーゲン線維性タンパク質の分泌によるものであり、コラーゲン線維性タンパク質を分泌した細胞はシャーレの底に接着した。
シャーレは予備加温したリン酸緩衝生理食塩水（ＰＢＳ）（Wako Pure Chemical Industries, Ltd.）で静かに３回すすぎ、非接着細胞を除去した。残っているｈＡＤＳＣｓは１０％ ＦＢＳ及び１％ Ｐ／Ｓを加えたＤＭＥＭ中で培養し（非特許文献３３）、そしてそれらがシャーレの面積の９０％を覆うように増殖するまで継代培養した。継代３代目のｈＡＤＳＣｓを以降の実験で使用した。It was possible to observe with an optical microscope that the morphology of the activated hADSCs changed from a circular shape to a spindle shape after the incubation for 1 day. These morphological changes were due to the secretion of collagen fibrillar protein from the cells, and the cells secreting the collagen fibrillar protein adhered to the bottom of the petri dish.
The petri dish was gently rinsed 3 times with pre-warmed phosphate buffered saline (PBS) (Wako Pure Chemical Industries, Ltd.) to remove non-adherent cells. The remaining hADSCs were cultured in DMEM supplemented with 10% FBS and 1% P / S (Non-patent Document 33) and subcultured until they grew to cover 90% of the petri dish area. The third passage of hADSCs was used in subsequent experiments.

（ｈＡＤＳＣｓの骨芽細胞への分化の誘導）
継代３代目のｈＡＤＳＣｓが骨形成への分化能を維持しているか分析した。詳しくは、分化誘導のために、ｈＡＤＳＣｓを公知の骨誘導培地で培養した。骨誘導培地は、１０％ ＦＢＳ、０．１μＭ デキサメタゾン、５０μＭ アスコルビン酸−２−リン酸、１０ｍＭ β−グリセロホスフェート、１％ Ｐ／Ｓを含むα−ＭＥＭ（Wako Pure Chemical Industries社）から成る（非特許文献６、３４−３７）。
１０％ ＦＢＳ及び１％ Ｐ／Ｓのみを含むα−ＭＥＭ溶液を、各条件の対照培地として使用した。
以下、ｈＡＤＳＣｓを骨誘導培地で分化誘導した細胞群をｈＡＤＳＣｓ誘導群と称し、ｈＡＤＳＣｓ対照培地で培養した細胞群をｈＡＤＳＣｓ対照群と称することがある。(Induction of differentiation of hADSCs into osteoblasts)
It was analyzed whether the third generation of hADSCs maintained the ability to differentiate into bone formation. Specifically, hADSCs were cultured in a known osteoinduction medium for differentiation induction. The osteoinduction medium consists of α-MEM (Wako Pure Chemical Industries) containing 10% FBS, 0.1 μM dexamethasone, 50 μM ascorbic acid-2-phosphate, 10 mM β-glycerophosphate, 1% P / S (non-Wako Pure Chemical Industries). Patent Document 6, 34-37).
An α-MEM solution containing only 10% FBS and 1% P / S was used as a control medium for each condition.
Hereinafter, a group of cells in which differentiation of hADSCs is induced in an osteoinduction medium may be referred to as a hADSCs induction group, and a group of cells cultured in a hADSCs control medium may be referred to as a hADSCs control group.

（ｈＡＤＳＣｓの骨芽細胞への分化誘導の確認）
ｈＡＤＳＣｓからの骨芽細胞分化誘導の成否を、アルカリホスファターゼ染色及びアリザリンレッド染色による組織化学分析により検討した。
詳しくは、アルカリホスファターゼ染色は、骨芽細胞機能の亢進及び骨形成活性亢進の指標として使用されている。アリザリンレッド染色は石灰化の促進の指標として使用されている。したがって、これら染色により陽性が確認された細胞は、骨芽細胞あるいは骨細胞としての機能を有すると考えることができる。(Confirmation of differentiation induction of hADSCs into osteoblasts)
The success or failure of osteoblast differentiation induction from hADSCs was examined by histochemical analysis with alkaline phosphatase staining and alizarin red staining.
Specifically, alkaline phosphatase staining is used as an indicator of increased osteoblast function and increased osteogenic activity. Alizarin red staining is used as an indicator of accelerated calcification. Therefore, the cells confirmed to be positive by these staining can be considered to have functions as osteoblasts or bone cells.

これら組織化学分析（非特許文献３８―４０）は、分化誘導培養期間中の１日目、３日目、５日目、７日目、１０日目、２週間目及び３週間目に実施した。   These histochemical analyzes (Non-Patent Documents 38-40) were performed on the 1st day, 3rd day, 5th day, 7th day, 10th day, 2nd week and 3rd week during the differentiation induction culture period. .

アルカリホスファターゼ染色実施時、培地は除去し、細胞層をＰＢＳで３回すすぎ、そして４％ パラホルムアルデヒド−リン酸緩衝液（Wako Pure Chemical Industries, Ltd）で室温にて５分間固定した。
その後、細胞層を脱イオン水で洗浄した。次いで、固定した細胞を1-Step NBT/BCIP plus Suppressor Solution（Thermo Fisher Scientific社）でインキュベーションした。３７℃で３０分間インキュベーション後、細胞層を脱イオン水で洗浄し、そして裸眼及び光学顕微鏡の両方で観察した。At the time of alkaline phosphatase staining, the medium was removed, the cell layer was rinsed 3 times with PBS, and fixed with 4% paraformaldehyde-phosphate buffer (Wako Pure Chemical Industries, Ltd) for 5 minutes at room temperature.
Thereafter, the cell layer was washed with deionized water. The fixed cells were then incubated with 1-Step NBT / BCIP plus Suppressor Solution (Thermo Fisher Scientific). After incubation at 37 ° C. for 30 minutes, the cell layer was washed with deionized water and observed with both the naked eye and light microscope.

アリザリンレッド染色の前に、細胞層をＰＢＳで３回すすぎ、そして４％ パラホルムアルデヒド−リン酸緩衝液（Wako Pure Chemical Industries社）で室温にて１０分間固定した。その後、細胞層を脱イオン水で３回洗浄した。次いで、アリザリンレッド染色をオステオジェネシスアッセイキット（ECM815, Millipore社）を使用して、標準プロトコルにしたがって行った（非特許文献４１）。   Prior to alizarin red staining, cell layers were rinsed 3 times with PBS and fixed with 4% paraformaldehyde-phosphate buffer (Wako Pure Chemical Industries) for 10 minutes at room temperature. The cell layer was then washed 3 times with deionized water. Subsequently, alizarin red staining was performed using an osteogenesis assay kit (ECM815, Millipore) according to a standard protocol (Non-patent Document 41).

（本発明のｈＡＤＳＣｓシートの作製）
継代３代目のｈＡＤＳＣｓを、１０ｃｍ シャーレに１×１０６細胞／シャーレで播種し、１０％ ＦＢＳ及び１％ Ｐ／Ｓを添加したＤＭＥＭ中でオーバーコンフルエントになるまで培養し、ｈＡＤＳＣｓシートを作製した。
作製培地は、１０％ ＦＢＳ、５０μＭ アスコルビン酸−２−リン酸、１％ Ｐ／Ｓを含むものである。培地は１週間の間、３日毎に交換した。これが、本願においてＡＤＳＣｓシートを作製するために開発した重要工程である。ｈＡＤＳＣｓシートが作成されたかどうかを確認するために、試料を無作為に採取して検査した。(Preparation of hADSCs sheet of the present invention)
The 3rd generation hADSCs were seeded in a 10 cm dish at 1 × 10 6 cells / dishlet and cultured in DMEM supplemented with 10% FBS and 1% P / S to prepare an hADSCs sheet. .
The production medium contains 10% FBS, 50 μM ascorbic acid-2-phosphate, 1% P / S. The medium was changed every 3 days for 1 week. This is an important process developed to produce ADSCs sheets in the present application. Samples were randomly taken and examined to see if hADSCs sheets were created.

（作製した細胞シートの骨芽細胞への分化の誘導）
ｈＡＤＳＣｓシートの骨分化を誘導するために、ｈＡＤＳＣｓシートを公知の誘導培地で培養した。
骨誘導培地は１０％ 、０．１μＭ デキサメタゾン、５０μＭ アスコルビン酸−２−リン酸、１０ｍＭ β−グリセロホスフェート、１％ Ｐ／Ｓを含むα−ＭＥＭから成る。
１０％ ＦＢＳ及び１％ Ｐ／Ｓのみを含むα−ＭＥＭ溶液を、各条件の対照培地として使用した。
以下、ｈＡＤＳＣｓシートを骨誘導培地で分化誘導した細胞群をｈＡＤＳＣｓシート誘導群と称し、ｈＡＤＳＣｓシートを対照培地で培養した細胞群をｈＡＤＳＣｓシート対照群と称することがある。(Induction of differentiation of the produced cell sheet into osteoblasts)
In order to induce bone differentiation of the hADSCs sheet, the hADSCs sheet was cultured in a known induction medium.
The osteoinduction medium consists of α-MEM containing 10%, 0.1 μM dexamethasone, 50 μM ascorbic acid-2-phosphate, 10 mM β-glycerophosphate, 1% P / S.
An α-MEM solution containing only 10% FBS and 1% P / S was used as a control medium for each condition.
Hereinafter, a cell group in which the hADSCs sheet is differentiated with the osteoinduction medium may be referred to as a hADSCs sheet induction group, and a cell group in which the hADSCs sheet is cultured in a control medium may be referred to as a hADSCs sheet control group.

（ｈＡＤＳＣｓシートのＡＬＰ染色キット及びアリザリンレッド染色キットによる染色）
ｈＡＤＳＣｓシートからの骨芽細胞分化誘導の成否を、アルカリホスファターゼ染色及びアリザリンレッド染色による組織化学分析により検討した。組織化学分析は、誘導培養期間中の１日目、３日目、５日目、７日目、１０日目、２週間目及び３週間目に実施した。ＡＬＰ染色及びアリザリンレッド染色は、上記した方法により実施した。(Staining of hADSCs sheet with ALP staining kit and alizarin red staining kit)
The success or failure of the induction of osteoblast differentiation from the hADSCs sheet was examined by histochemical analysis using alkaline phosphatase staining and alizarin red staining. Histochemical analysis was performed on days 1, 3, 5, 7, 10, 10, 2 and 3 during the induction culture period. ALP staining and alizarin red staining were performed by the methods described above.

（ＡＬＰ活性の定量及び統計解析）
ｈＡＤＳＣｓ対照群、ｈＡＤＳＣｓ誘導群、ｈＡＤＳＣｓシート対照群及びｈＡＤＳＣｓシート誘導群のそれぞれについてＡＬＰ活性を測定した。
ＡＬＰ活性の測定はTRACP & ALP assay kit（MK301、TaKaRa Bio社）を使用して製造者使用説明書にしたがって行った。チェックポイントは、培養期間の１日目、３日目、５日目、７日目、１０日目、２週間目及び３週間目に設定した。上記４群間のＡＬＰ活性の差異の有意性は、スチューデントｔ−検定を使用した統計解析により評価した。ｐ値が０．０５以下のとき有意であると判定した。(Quantification and statistical analysis of ALP activity)
ALP activity was measured for each of the hADSCs control group, the hADSCs induction group, the hADSCs sheet control group, and the hADSCs sheet induction group.
The ALP activity was measured using a TRACP & ALP assay kit (MK301, TaKaRa Bio) according to the manufacturer's instructions. Checkpoints were set on the 1st, 3rd, 5th, 7th, 10th, 2nd and 3rd weeks of the culture period. The significance of the difference in ALP activity between the four groups was evaluated by statistical analysis using Student's t-test. The p value was determined to be significant when it was 0.05 or less.

（結果）
ｈＡＤＳＣｓ及びｈＡＤＳＣｓシートを光学顕微鏡で観察した。そして、細胞形態の変化を、実験期間中、観察した。細胞数はオーバーコンフルエント状態になった後には変化しなかった。ｈＡＤＳＣｓシートの細胞形態がオーバーコンフルエント状態のｈＡＤＳＣｓのものと同じであることが確認できた（図１）。(result)
hADSCs and hADSCs sheets were observed with an optical microscope. Changes in cell morphology were then observed during the experiment. The cell number did not change after becoming overconfluent. It was confirmed that the cell morphology of the hADSCs sheet was the same as that of the hADSCs in an overconfluent state (FIG. 1).

ｈＡＤＳＣｓ対照群とｈＡＤＳＣｓ誘導群との間のＡＬＰ染色の結果の差異は、誘導開始後２週間目に認められ、そしてこれら２群間のアリザリンレッド染色の結果の差異は３週間目に認められた（図２）。
これに対し、ｈＡＤＳＣｓシート対照群とｈＡＤＳＣｓシート誘導群との間の差異はＡＬＰ染色では５日目に、そしてアリザリンレッド染色では７日目に認められた（図３）。A difference in the results of ALP staining between the hADSCs control group and the hADSCs induction group was observed 2 weeks after initiation of induction, and a difference in the results of alizarin red staining between these 2 groups was observed at 3 weeks. (FIG. 2).
In contrast, a difference between the hADSCs sheet control group and the hADSCs sheet induction group was found on day 5 for ALP staining and on day 7 for alizarin red staining (FIG. 3).

ＡＬＰ染色及びアリザリンレッド染色のいずれにおいても、その染色濃度は経時的に濃くなった。それぞれ対応する染色チェックポイントで、ｈＡＤＳＣｓシート誘導群のＡＬＰ染色及びアリザリンレッド染色の濃度は、ｈＡＤＳＣｓ誘導群のものより濃かった（図２、３）。   In both ALP staining and alizarin red staining, the staining concentration increased with time. At each corresponding staining checkpoint, the concentration of ALP staining and alizarin red staining in the hADSCs sheet induction group was higher than that in the hADSCs induction group (FIGS. 2 and 3).

ｈＡＤＳＣｓ誘導群のＡＬＰ活性の平均値は、ｈＡＤＳＣｓのＡＬＰ活性が骨誘導の５日後から急増したことを示す（図４）。Life Technologies社の使用者説明書と一致して、この増加は１４日目に最高値に達し、他の研究による報告と一致する（非特許文献４２）。ｈＡＤＳＣｓ対照群のＡＬＰ活性は、実験期間全体にわたって低値の範囲にとどまっていた。   The average value of ALP activity in the hADSCs induction group shows that the ALP activity of hADSCs increased rapidly from 5 days after bone induction (FIG. 4). Consistent with Life Technologies user instructions, this increase reached a maximum on day 14 and is consistent with reports from other studies (42). The ALP activity in the hADSCs control group remained in the low range throughout the experimental period.

ｈＡＤＳＣｓシート誘導群及びｈＡＤＳＣｓシート対照群はいずれも１日目に、ｈＡＤＳＣｓ誘導群及びｈＡＤＳＣｓ対照群とそれぞれ比較して高いＡＬＰ活性を示した（Ｐ＜０．０５）。特に、ｈＡＤＳＣｓシート誘導群は、ｈＡＤＳＣｓ誘導群より極めて高い値を示した（Ｐ＜０．０５）。ｈＡＤＳＣｓシート誘導群のＡＬＰ活性の直線的増加は１日目から始まり、そして１０日目に最高近くに達した。しかしながら、ｈＡＤＳＣｓシート誘導群のＡＬＰ活性は１４日目及び２１日目でも未だ増加していた。ｈＡＤＳＣｓシート対照群のＡＬＰ活性は、ｈＡＤＳＣｓシート誘導群と比較して、実験期間全体にわたって低値の範囲にとどまっていたが、その値はｈＡＤＳＣｓ対照群のものよりずっと大きかった（Ｐ＜０．０５）（図４）。   Both the hADSCs sheet induction group and the hADSCs sheet control group showed higher ALP activity on the first day compared with the hADSCs induction group and the hADSCs control group, respectively (P <0.05). In particular, the hADSCs sheet induction group showed a significantly higher value than the hADSCs induction group (P <0.05). The linear increase in ALP activity in the hADSCs sheet-induced group began on day 1 and reached a maximum on day 10. However, the ALP activity in the hADSCs sheet-induced group was still increased on the 14th and 21st days. The ALP activity of the hADSCs sheet control group remained in the low range over the entire experimental period compared to the hADSCs sheet induction group, but the value was much greater than that of the hADSCs control group (P <0.05). (FIG. 4).

このように、ＡＤＳＣｓシート誘導群のＡＬＰ活性が、各時点で分散ＡＤＳＣｓ群のものより高かったことから、ＡＤＳＣｓシートが分散ＡＤＳＣｓと比較して骨形成により有効かつ迅速に作用することが確認された。
すなわち、本発明のＡＤＳＣｓシートの移植片は移植後に、個々別々の分散細胞よりもより早く骨形成にその役割を果たすことができるという利点を有するため、臨床治療に有用である。Thus, since the ALP activity of the ADSCs sheet induction group was higher than that of the dispersed ADSCs group at each time point, it was confirmed that the ADSCs sheet acts more effectively and rapidly on bone formation than the dispersed ADSCs. .
That is, the ADSCs sheet graft of the present invention is useful for clinical treatment because it has the advantage that it can play its role in bone formation sooner after transplantation than individual discrete dispersed cells.

（添加するアスコルビン酸濃度の最適化）
上記実施例１において、骨分化能を有する脂肪幹細胞シート及び骨芽細胞を含む細胞シートは優れた骨分化誘導能を有することを確認した。さらに、本実施例では、最適な添加するアスコルビン酸濃度を検討した。詳細は、以下の通りである。(Optimization of added ascorbic acid concentration)
In Example 1 above, it was confirmed that the adipose stem cell sheet having the ability to differentiate into bone and the cell sheet containing osteoblasts have an excellent ability to induce bone differentiation. Further, in this example, the optimum concentration of ascorbic acid to be added was examined. Details are as follows.

（ＡＬＰ活性の確認）
５０ μＭ、１５０ μＭ又は４５０ μＭのアスコルビン酸濃度の培地条件で作製したＡＤＳＣｓシートのＡＬＰ活性を４０５ ｎｍ波長の吸光度により測定した。１５０ μＭアスコルビン酸含有培地で作製したＡＤＳＣｓシートは、５０ μＭ及び４５０ μＭのアスコルビン酸含有培地で作製したＡＤＳＣｓシートと比較して、ＡＬＰ活性が高かった（参照：図５）。
これにより、ＡＤＳＣｓシート培地のアスコルビン酸濃度の最適な範囲は、５０ μＭ〜５００ μＭの範囲、より好ましくは１００ μＭ〜２００ μＭであることを確認した。
なお、アスコルビン酸濃度５０ μＭでは、ＡＤＳＣｓシート作製に通常７日間かかる。しかし、アスコルビン酸濃度１５０ μＭでは、ＡＤＳＣｓシート作製は約５日間から作製できた。(Confirmation of ALP activity)
The ALP activity of ADSCs sheets prepared under medium conditions of 50 μM, 150 μM or 450 μM ascorbic acid concentration was measured by absorbance at 405 nm wavelength. ADSCs sheets prepared with 150 μM ascorbic acid-containing medium had higher ALP activity than ADSCs sheets prepared with 50 μM and 450 μM ascorbic acid-containing media (see FIG. 5).
Thereby, it was confirmed that the optimal range of the ascorbic acid concentration of the ADSCs sheet medium is in the range of 50 μM to 500 μM, more preferably 100 μM to 200 μM.
When the ascorbic acid concentration is 50 μM, ADSCs sheet production usually takes 7 days. However, at an ascorbic acid concentration of 150 μM, ADSCs sheets could be produced from about 5 days.

（ＡＤＳＣｓシートの強度の確認）
各培養日数において、ＡＤＳＣｓシートの強度を確認した。５０ μＭ、１５０μＭ及び４５０ μＭのアスコルビン酸濃度のいずれの場合においても、力学的強度を有しかつ破れにくいことを確認した。特に、５０ μＭアスコルビン酸濃度ではスクレーパーでゆっくりそっと剥ぎ取らなければならないのに対して、１５０ μＭ及び４５０ μＭアスコルビン酸濃度ではピンセット（トゥイザーズ）でつまんで持ち上げることができた。すなわち、アスコルビン酸濃度１５０ μＭ及び４５０ μＭでは、アスコルビン酸濃度５０ μＭと比較して、優れた力学的強度を有していることを確認した。
これにより、ＡＤＳＣｓシートが力学的強度を有しかつ自身単独で骨修復部位に固定されるので、スキャホールドを必要としないことを確認した。(Confirmation of ADSCs sheet strength)
In each culture day, the strength of the ADSCs sheet was confirmed. It was confirmed that each of the ascorbic acid concentrations of 50 μM, 150 μM and 450 μM had mechanical strength and was not easily torn. In particular, the 50 μM ascorbic acid concentration had to be gently peeled off with a scraper, whereas the 150 μM and 450 μM ascorbic acid concentrations could be picked up with tweezers (twisers). That is, it was confirmed that the ascorbic acid concentrations of 150 μM and 450 μM have excellent mechanical strength compared to the ascorbic acid concentration of 50 μM.
This confirmed that the ADSCs sheet has mechanical strength and is fixed to the bone repair site by itself, so that no scaffold is required.

（総論）
本発明のＡＤＳＣｓシートの骨分化が早いのは、コラーゲン分泌が高いためであると考えられる。アスコルビン酸−２−リン酸は、間葉系細胞のコラーゲンタンパク質分泌を増加させることが報告されており（非特許文献３２）、また、コラーゲンタンパク質は骨形成系列において細胞シートの良好な天然担体と考えられている（非特許文献３１）。
本発明のＡＤＳＣｓシートは、コラーゲンタンパク質の分泌のゆえに、著しい力学的強度を示し、それらは従来の実験用ピンセット及び従来の外科用具類で直接持つことができる（非特許文献４３、４４）という利点を有する。この点からも、本発明のＡＤＳＣｓシートはインビトロ・インビボでの骨再生及び骨再建に高い有効性を有する。(General)
It is considered that the bone differentiation of the ADSCs sheet of the present invention is fast because collagen secretion is high. Ascorbic acid-2-phosphate has been reported to increase collagen protein secretion in mesenchymal cells (Non-patent Document 32), and collagen protein is a natural carrier that has a good cell sheet in the osteogenic series. It is considered (Non-Patent Document 31).
The ADSCs sheets of the present invention exhibit significant mechanical strength due to the secretion of collagen proteins, and they can be directly held by conventional laboratory tweezers and conventional surgical instruments (Non-patent Documents 43 and 44). Have Also from this point, the ADSCs sheet of the present invention has high effectiveness for bone regeneration and bone reconstruction in vitro and in vivo.

本発明は、骨組織再生分野、例えば整形外科分野において、骨再生及び骨再建のための材料及びその迅速で有効な作製方法を提供するものであり、このような分野において極めて有用である。   The present invention provides a material for bone regeneration and bone reconstruction and a rapid and effective production method thereof in the field of bone tissue regeneration, for example, orthopedics, and is extremely useful in such a field.

Claims (8)

細胞分化誘導剤を含まない細胞培養培地にアスコルビン酸又はその塩を添加して調製した溶液を、スキャホールドを添加していない培養容器内に加え、該溶液中で、脂肪由来幹細胞を培養することを含む、骨分化能を有する脂肪由来幹細胞シートの作製方法。   A solution prepared by adding ascorbic acid or a salt thereof to a cell culture medium not containing a cell differentiation inducer is added to a culture vessel to which no scaffold is added, and adipose-derived stem cells are cultured in the solution. A method for producing an adipose-derived stem cell sheet having bone differentiation ability. 前記アスコルビン酸又はその塩が、５０μＭ〜５００μＭの濃度になるように細胞培養培地に添加されることを特徴とする、請求項１に記載の作製方法。   The production method according to claim 1, wherein the ascorbic acid or a salt thereof is added to the cell culture medium so as to have a concentration of 50 μM to 500 μM. 前記脂肪由来幹細胞を培養することが、脂肪由来幹細胞を３日〜１５日間培養することである、請求項１又は２に記載の作製方法。   The production method according to claim 1 or 2, wherein culturing the adipose-derived stem cells is culturing the adipose-derived stem cells for 3 to 15 days. 前記脂肪由来幹細胞シートが単層の脂肪由来幹細胞シートである、請求項１〜３のいずれか１に記載の作製方法。   The production method according to any one of claims 1 to 3, wherein the fat-derived stem cell sheet is a monolayer fat-derived stem cell sheet. 請求項１〜４のいずれか１項に記載の作製方法により製造された、骨分化能を有する脂肪幹細胞シート。   The adipose stem cell sheet | seat which has the bone differentiation ability manufactured by the preparation method of any one of Claims 1-4. 請求項５に記載の脂肪幹細胞シートを、骨分化誘導剤を含む細胞培養培地中で培養することを含む、骨芽細胞を含む細胞シートの作製方法。   A method for producing a cell sheet containing osteoblasts, comprising culturing the adipose stem cell sheet according to claim 5 in a cell culture medium containing a bone differentiation-inducing agent. 請求項５に記載の骨分化能を有する脂肪幹細胞シート又は請求項６に記載の作製方法により製造された骨芽細胞を含む細胞シートを含む骨組織再生用材料。   A material for regenerating bone tissue comprising the adipose stem cell sheet having the ability to differentiate into bone according to claim 5 or the cell sheet containing osteoblasts produced by the production method according to claim 6. 前記骨組織再生用材料は、スキャホールドを含まないことを特徴とする、請求項７に記載の骨組織再生用材料。   The bone tissue regeneration material according to claim 7, wherein the bone tissue regeneration material does not include a scaffold.
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