JP6338259B1 - Meniscus regeneration material and method for producing meniscus regeneration material - Google Patents
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Abstract
【課題】半月板の再生をより促進することが可能な半月板再生用材料を提供する。【解決手段】膝関節の半月板の少なくとも一部を再生するため、膝関節内に移植されるように用いられる半月板再生用材料１０である。半月板再生用材料１０は、コラーゲンスポンジ１１と、コラーゲンスポンジに含浸された脂肪組織由来再生細胞（ＡＤＲＣｓ）１２と、を有する。コラーゲンスポンジ１１は、アテロコラーゲンスポンジであることが望ましい。コラーゲンスポンジ１１には、脂肪組織由来再生細胞とアテロコラーゲンゲルとが混合されたＡＤＲＣｓ溶液１３を含浸させることが望ましい。【選択図】図１A meniscus regeneration material capable of further promoting the regeneration of a meniscus. A meniscal regeneration material that is used to be implanted into a knee joint to regenerate at least a portion of the meniscus of a knee joint. The meniscal regeneration material 10 includes a collagen sponge 11 and adipose tissue-derived regenerative cells (ADRCs) 12 impregnated in the collagen sponge. The collagen sponge 11 is desirably an atelocollagen sponge. The collagen sponge 11 is preferably impregnated with an ADRCs solution 13 in which adipose tissue-derived regenerative cells and atelocollagen gel are mixed. [Selection] Figure 1
Description
本発明は、半月板再生用材料及び半月板再生用材料の作製方法に関する。 The present invention relates to a meniscus regeneration material and a method for producing a meniscus regeneration material .
半月板は、膝関節内にある軟骨様組織であり、スポーツ活動や加齢などにより損傷・断裂を生じて半月板損傷を起こすことがある。半月板損傷は、慢性化すると痛みを伴う変形性膝関節症に移行するため早期の治療が必要である。半月板損傷の治療には、一般的に切除術、縫合術、保存療法などあるが、半月板内側は血管がなく、自然治癒は困難なことから、再生治療に関する技術の開発が望まれている。 The meniscus is a cartilage-like tissue in the knee joint, and may cause meniscus damage due to injury or tear caused by sports activities or aging. When meniscus injury becomes chronic, it shifts to painful knee osteoarthritis, so early treatment is necessary. Treatment of meniscal injury generally includes excision, suturing, and conservative therapy. However, since there is no blood vessel inside the meniscus and natural healing is difficult, development of technology related to regenerative treatment is desired. .
このような再生治療に関する技術として、例えば、特許文献１には、自己再生能と多分化能を有する間葉幹細胞（ＭＳＣｓ）を、半月板欠損部を覆うように該欠損部に注入することで、軟骨組織を再生させる技術が開示されている。 As a technique related to such regenerative treatment, for example, Patent Document 1 discloses that mesenchymal stem cells (MSCs) having self-renewal ability and pluripotency are injected into the defect part so as to cover the meniscus defect part. A technique for regenerating cartilage tissue is disclosed.
また、特許文献２には、架橋コラーゲンスポンジと膝蓋下脂肪体とからなる半月板再生基材が開示されている。この基材を膝関節の半月板の欠損部分に充填することで、膝蓋下脂肪体に含まれる間葉系幹細胞により半月板の再生を促進しようとするものである。 Patent Document 2 discloses a meniscal regeneration base material composed of a crosslinked collagen sponge and a subpatellar fat body. By filling this base material into the meniscus defect of the knee joint, the mesenchymal stem cells contained in the subpatellar fat body are intended to promote the regeneration of the meniscus.
しかしながら、特許文献１に記載の技術では、間葉幹細胞を直に体内に注入するため、間葉幹細胞が欠損部に滞留しにくく、体内に吸収されて十分な再生が行われないおそれがある。また、治療に充分な間葉系幹細胞を取得するためには長期間の培養が必要であるとともに、間葉系幹細胞を採取と移植のために複数回の手術をする必要があり、患者への負担が大きい。 However, in the technique described in Patent Document 1, since mesenchymal stem cells are directly injected into the body, the mesenchymal stem cells are unlikely to stay in the defect and may not be sufficiently regenerated by being absorbed into the body. In addition, long-term culture is required to obtain mesenchymal stem cells sufficient for treatment, and it is necessary to perform multiple operations for collecting and transplanting mesenchymal stem cells. The burden is heavy.
また、特許文献２に記載の基材でも、架橋コラーゲンスポンジと膝蓋下脂肪体とが積層されているか、または架橋コラーゲンスポンジが膝蓋下脂肪体に包埋されている構成であるため、膝蓋下脂肪体が半月板近傍に滞留しにくく、再生が起こる前に体内に吸収されてしまう可能性がある。また、患者から採取した膝蓋下脂肪体をそのまま用いるため、再生に関与する間葉系幹細胞の量も少なく、再生が十分に行えない可能性があり、改良の余地がある。 Further, even in the base material described in Patent Document 2, since the cross-linked collagen sponge and the sub-patellar fat pad are laminated or the cross-linked collagen sponge is embedded in the sub-patellar fat pad, The body is less likely to stay near the meniscus and may be absorbed by the body before regeneration occurs. Moreover, since the subpatellar fat pad collected from the patient is used as it is, the amount of mesenchymal stem cells involved in the regeneration is small, and there is a possibility that regeneration cannot be performed sufficiently, and there is room for improvement.
本発明は、上記の事情に鑑みて為されたもので、半月板の再生をより促進することが可能な半月板再生用材料を提供することを目的とする。 This invention is made | formed in view of said situation, and it aims at providing the material for meniscus reproduction | regeneration which can accelerate | stimulate the reproduction | regeneration of a meniscus more.
上記の目的を達成するため、本願に係る半月板再生用材料は、膝関節の半月板の少なくとも一部を再生するため、前記膝関節内に移植されるように用いられる半月板再生用材料であって、コラーゲンスポンジと、前記コラーゲンスポンジに含浸された脂肪組織由来再生細胞と、を有し、前記脂肪組織由来再生細胞は、生体内から採取された脂肪組織から分離され培養されることなく得られた、脂肪組織由来幹細胞を含む細胞集団であることを特徴とする。 In order to achieve the above object, the meniscus regeneration material according to the present application is a material for meniscus regeneration used to be transplanted into the knee joint in order to regenerate at least part of the meniscus of the knee joint. there, possess a collagen sponge, and fat tissue derived regenerative cells impregnated before Symbol collagen sponge, wherein the adipose tissue-derived regenerative cells, without being separated from adipose tissue collected from a living body are cultured The obtained cell population includes adipose tissue-derived stem cells .
本発明によれば、半月板の再生をより促進することが可能な半月板再生用材料を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the meniscus reproduction | regeneration material which can accelerate | stimulate the reproduction | regeneration of a meniscus more can be provided.
近年、脂肪組織由来幹細胞や、それらを含む細胞集団である脂肪組織由来再生細胞（ＡＤＲＣｓ、以下、単に「ＡＤＲＣｓ」ということがある。）を利用した組織再構築が臨床応用に向けて研究されている。本願の発明者は、半月板損傷治療の新規細胞ソースとして、自己再生能と多分化能に優れるＡＤＲＣｓに着目し、本願発明をするに至った。 In recent years, tissue reconstruction using adipose tissue-derived stem cells and adipose tissue-derived regenerative cells (ADRCs, hereinafter simply referred to as “ADRCs”), which is a cell population containing them, has been studied for clinical application. Yes. The inventor of the present application has focused on ADRCs excellent in self-renewal ability and pluripotency as a novel cell source for meniscal injury treatment, and has come to make the present invention.
以下、本願の一実施形態に係る半月板再生用材料について、図面を参照しながら説明する。図１は、本実施形態の半月板再生用材料の概略図である。この図１に示す本実施形態の半月板再生用材料１０は、膝関節の半月板の少なくとも一部を再生するため、膝関節内に移植されるように用いられる。この半月板再生用材料１０は、コラーゲンスポンジ１１と、コラーゲンスポンジ１１に含浸された脂肪組織由来再生細胞（ＡＤＲＣｓ）（図１に符号「１２」で示した）と、を有して構成される。 Hereinafter, a meniscal regeneration material according to an embodiment of the present application will be described with reference to the drawings. FIG. 1 is a schematic view of the meniscal regeneration material of the present embodiment. The meniscus regeneration material 10 of this embodiment shown in FIG. 1 is used so as to be transplanted into the knee joint in order to regenerate at least a part of the meniscus of the knee joint. The meniscal regeneration material 10 includes a collagen sponge 11 and adipose tissue-derived regenerative cells (ADRCs) impregnated in the collagen sponge 11 (indicated by reference numeral “12” in FIG. 1). .
また、本実施形態では、ＡＤＲＣｓ１２とコラーゲンゲルとの混合溶液であるＡＤＲＣｓ溶液１３を用いることで、コラーゲンスポンジ１１に含浸させ易くしている。図２の膝関節１の断面図に示されるように、半月板２は、膝関節１の内側と外側に対向するように形成された内側半月板２ａと外側半月板２ｂとからなる。半月板２は、線維軟骨およびコラーゲンから構成される組織であるため、本実施形態のようなコラーゲンスポンジ１１、コラーゲンゲルを用いることで、組成物としても違和感のない使用が可能であり、拒絶反応等を抑制することができる。 In the present embodiment, the collagen sponge 11 is easily impregnated by using the ADRCs solution 13 which is a mixed solution of ADRCs 12 and collagen gel. As shown in the sectional view of the knee joint 1 in FIG. 2, the meniscus 2 includes an inner meniscus 2 a and an outer meniscus 2 b formed so as to face the inside and the outside of the knee joint 1. Since the meniscus 2 is a tissue composed of fibrocartilage and collagen, the use of the collagen sponge 11 and the collagen gel as in the present embodiment makes it possible to use the composition without any sense of incongruity, and rejection. Etc. can be suppressed.
上述のような構成の半月板再生用材料１０を膝関節の半月板の欠損部に移植すると、物質の保持性に優れるコラーゲンスポンジ１１によって、ＡＤＲＣｓが流出して体内に吸収されるのを良好に抑制することができる。そして、自己再生能と多分化能に優れるＡＤＲＣｓが、コラーゲンスポンジ１１を骨格として増殖することで、半月板の再生を、より効果的に促進することが可能となる。 When the meniscus regeneration material 10 having the above-described configuration is transplanted into the meniscus defect of the knee joint, the collagen sponge 11 having excellent substance retention improves the ability of ADRCs to flow out and be absorbed into the body. Can be suppressed. And ADRCs which are excellent in self-regenerative ability and multipotency can proliferate meniscus more effectively by proliferating using collagen sponge 11 as a skeleton.
また、特許文献２で使用する膝蓋下脂肪体は、体内での分量がもともと少ないため、十分な分量の幹細胞を得るのは困難であるとともに、採取の際に身体へ負担がかかる。これに対して、本実施形態で使用するＡＤＲＣｓは、体内に多く含まれる脂肪組織から採取できるため、再生に十分な分量のＡＤＲＣｓを容易かつ、低侵襲で採取することができる。 In addition, since the subpatellar fat pad used in Patent Document 2 is originally small in the body, it is difficult to obtain a sufficient amount of stem cells, and a burden is imposed on the body during collection. On the other hand, since ADRCs used in the present embodiment can be collected from adipose tissue contained in a large amount in the body, an amount of ADRCs sufficient for regeneration can be collected easily and with minimal invasiveness.
また、コラーゲンスポンジ１１のみの移植では、組織の再生が十分に行われない。本実施形態の半月板再生用材料１０のように、コラーゲンスポンジ１１とＡＤＲＣｓとの組み合わせで、しかも、ＡＤＲＣｓをコラーゲンスポンジ１１に含浸させることで、半月板の再生を顕著に促進することができるものである。 In addition, transplantation with only the collagen sponge 11 does not sufficiently regenerate the tissue. Like the meniscus regeneration material 10 of this embodiment, the combination of the collagen sponge 11 and ADRCs, and the impregnation of the ADRCs with the collagen sponge 11 can remarkably promote meniscus regeneration. It is.
また、本実施形態では、ＡＤＲＣｓとコラーゲンゲルとを混合したＡＤＲＣｓ溶液１３を用いているため、ＡＤＲＣｓをコラーゲンスポンジ１１に含浸させ易くなる。さらに、コラーゲンスポンジ１１から体内へＡＤＲＣｓの流出防止効果が高まるとともに、コラーゲンゲルも足場として機能し、半月板の再生能力を、より向上させることができる。 Moreover, in this embodiment, since the ADRCs solution 13 which mixed ADRCs and collagen gel is used, it becomes easy to impregnate the ADRCs in the collagen sponge 11. Furthermore, the effect of preventing the outflow of ADRCs from the collagen sponge 11 into the body is enhanced, and the collagen gel also functions as a scaffold, so that the meniscus regeneration ability can be further improved.
また、コラーゲンスポンジとしては、ＡＤＲＣｓを含有できるものであれば、特に限定されないが、アテロコラーゲンを材料としたアテロコラーゲンスポンジを用いるのが最適である。また、ＡＤＲＣｓを混入させるコラーゲンゲルも、アテロコラーゲンを可溶化したものを用いることが好ましい。アテロコラーゲンは、アレルギーなどの原因となる抗原性を有するテロペプチドを酵素処理等で除去したコラーゲンであるため好ましい。 The collagen sponge is not particularly limited as long as it can contain ADRCs, but it is optimal to use an atelocollagen sponge made of atelocollagen. Moreover, it is preferable to use what solubilized atelocollagen as the collagen gel mixed with ADRCs. Atelocollagen is preferred because it is a collagen obtained by removing an antigenic telopeptide that causes allergies and the like by enzymatic treatment.
また、コラーゲンスポンジ１１へ含浸させるＡＤＲＣｓの細胞数が、１．７×１０4 cells/mm3以上であることが望ましい。このような細胞数のＡＤＲＣｓを含浸させたコラーゲンスポンジ１１からなる半月板再生用材料１０を移植することで、半月板の再生を、より効果的に促進することができる。このような半月板再生用材料１０を得るには、５×１０5 cells/10μL以上となるように調製したコラーゲンゲル溶液１３をコラーゲンスポンジ１１に含浸させることが望ましい。 Moreover, it is desirable that the number of cells of ADRCs impregnated in the collagen sponge 11 is 1.7 × 10 4 cells / mm 3 or more. By transplanting the meniscus regeneration material 10 made of the collagen sponge 11 impregnated with ADRCs having such a number of cells, regeneration of the meniscus can be promoted more effectively. In order to obtain such a meniscal regeneration material 10, it is desirable to impregnate collagen sponge 11 with collagen gel solution 13 prepared so as to be 5 × 10 5 cells / 10 μL or more.
以下に実施例を挙げて本発明の態様を更に詳しく説明するが、本発明はこれら実施例に限定されるものではない。 Hereinafter, embodiments of the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
（実施例１）
Ｉ型のアテロコラーゲンゲル（高研社製、ＡｔｅｌｏＣｅｌｌ ＩＰＣ−５０）を培地で中和した溶液１０μｌに、５×１０5個のＡＤＲＣｓを均一に懸濁し、ＡＤＲＣｓ溶液１３を得た。このＡＤＲＣｓ溶液１３を、１／４円の扇形、厚さ１／２に切断したアテロコラーゲンスポンジ（高研社製、ＭＩＧＨＴＹ、直径約５×厚み３ｍｍ）へ滴下した。滴下後に１，０００ｒｐｍで１分間遠心して、アテロコラーゲンスポンジ１１全体に、ＡＤＲＣｓが混入したアテロコラーゲンを浸透させて、実施例１の半月板再生用材料１０を作製した（図１、図４参照）。
Example 1
5 × 10 5 ADRCs were uniformly suspended in 10 μl of a neutralized medium of type I atelocollagen gel (manufactured by Koken Co., Ltd., AteloCell IPC-50) to obtain ADRCs solution 13. This ADRCs solution 13 was dropped onto an atelocollagen sponge (manufactured by Koken Co., Ltd., MIGHTY, diameter: about 5 × thickness: 3 mm) cut into a quarter-circle sector and a thickness of ½. After dropping, the mixture was centrifuged at 1,000 rpm for 1 minute, and the atelocollagen sponge 11 was infiltrated with atelocollagen mixed with ADRCs to produce the meniscal regeneration material 10 of Example 1 (see FIGS. 1 and 4).
（比較例１）
実施例１と同様のアテロコラーゲンスポンジに、アテロコラーゲンゲルのみを浸透させて、比較例１の半月板再生用材料２０を作製した（図４参照）。
(Comparative Example 1)
Only the atelocollagen gel was infiltrated into the atelocollagen sponge similar to that in Example 1 to prepare the meniscal regeneration material 20 of Comparative Example 1 (see FIG. 4).
（評価試験１）
評価試験１では、脂肪組織に含まれるＡＤＲＣｓの軟骨細胞と骨芽細胞への分化能について評価した。まず、８〜１０週齢の雄ＳＤラットの鼠蹊部の脂肪組織からＡＤＲＣｓを単離して、ＣＤ３１（血管内皮細胞），ＣＤ３４（幹細胞），ＣＤ４５（リンパ球）の発現をフローサイトメーターで解析した。
(Evaluation Test 1)
In Evaluation Test 1, the ability of ADRCs contained in adipose tissue to differentiate into chondrocytes and osteoblasts was evaluated. First, ADRCs were isolated from the adipose tissue of the buttock of 8-10 week old male SD rats, and the expression of CD31 (vascular endothelial cells), CD34 (stem cells), and CD45 (lymphocytes) was analyzed with a flow cytometer. .
次に、軟骨細胞誘導培地（10% FBS αMEM, 2 mM L-glutamine, 2 mM Sodium pyrnvate,10-4 M Ascolbicacid, 10 nM Dexamethasone, 1% ITS premix,10 ng/ml TGF-β3）と骨芽細胞誘導培地（10% FBS αMEM, 10 mMβ-glycerophosphate,50 μg/ml Ascolbicacid, 10-8 M Dexamethasone）で３週間培養した。培養後に、軟骨細胞誘導と、骨芽細胞への誘導と、石灰化能を評価した。評価方法として、軟骨細胞誘導は、Alcian Blue（アルシアンブルー）染色、Toluidine blue（トルイジンブルー）染色で評価し、骨芽細胞への誘導はalkaline phosphatase（アルカリホスファターゼ）（ＡＬＰ）活性染色で評価し、石灰化能はAlizarin red（アリザリンレッド）染色とvon Kossa（フォン・コッサ）染色で評価した。図３Ａ〜図３Ｃに、評価試験結果を示す。 Next, chondrocyte induction medium (10% FBS αMEM, 2 mM L-glutamine, 2 mM Sodium pyrnvate, 10 -4 M Ascolbicacid, 10 nM Dexamethasone, 1% ITS premix, 10 ng / ml TGF-β3) and osteoblast The cells were cultured in a cell induction medium (10% FBS αMEM, 10 mM β-glycerophosphate, 50 μg / ml Ascolbicacid, 10 −8 M Dexamethasone) for 3 weeks. After culture, chondrocyte induction, osteoblast induction, and calcification ability were evaluated. As evaluation methods, chondrocyte induction is evaluated by Alcian Blue staining and Toluidine blue staining, and induction into osteoblasts is evaluated by alkaline phosphatase (ALP) activity staining. The calcification ability was evaluated with Alizarin red staining and von Kossa staining. 3A to 3C show the evaluation test results.
本評価試験１において、ラットの脂肪組織１ｇあたり細胞数約１０６個が回収できた。図３Ａの（ａ）に示すように、脂肪組織由来幹細胞の指標であるＣＤ３１（−），ＣＤ３４（＋），ＣＤ４５（−）細胞は全細胞数のおよそ４．０％であった。一方、図３Ａの（ｂ）に示すように、骨髄細胞の中には、脂肪組織由来幹細胞の集団は認められなかった。 In this evaluation test 1, about 10 6 cells were recovered per 1 g of rat adipose tissue. As shown in (a) of FIG. 3A, CD31 (−), CD34 (+), and CD45 (−) cells, which are indicators of adipose tissue-derived stem cells, were approximately 4.0% of the total number of cells. On the other hand, as shown in FIG. 3A (b), a population of stem cells derived from adipose tissue was not observed in the bone marrow cells.
本評価試験１によれば、ＡＤＲＣｓは培養条件によって、軟骨細胞様細胞または骨芽細胞様細胞へ分化した。軟骨細胞誘導培地にて２１日培養後に各種染色法にて観察すると、図３Ｂのｃ、ｄにそれぞれ示すように、軟骨基質に含まれる酸性ムコ多糖を染めるアルシアンブルー染色とトルイジンブルー染色とは、ともに陽性細胞が認められた。図３Ｂのａ、ｂはコントロール培地（10% FBS αMEM）の染色結果を示す。 According to this evaluation test 1, ADRCs differentiated into chondrocyte-like cells or osteoblast-like cells depending on the culture conditions. When observed with various staining methods after 21 days of culturing in the chondrocyte induction medium, as shown in FIGS. 3B and 3C, the alcian blue staining and toluidine blue staining for staining acidic mucopolysaccharide contained in the cartilage matrix are shown. In both cases, positive cells were observed. 3B, a and b show the staining results of the control medium (10% FBS αMEM).
また、骨芽細胞誘導培地にて２１日培養後に各種染色法にて観察した。その結果を図３Ｃに示す。図３Ｃのａ，ｂ，ｃはコントロール培地、ｄ，ｅ，ｆは骨芽細胞誘導培地、ｇ，ｈ，ｉは骨芽細胞誘導培地＋ＢＭＰ-2 100 ng/mlの各染色結果を示す。この図３Ｃによれば、骨芽細胞分化の指標であるアルカリホスファターゼ活性染色陽性の細胞が増加して、石灰化の指標であるフォン・コッサ染色とアリザリンレッド染色陽性の領域が拡大した。 Moreover, it observed by various dyeing | staining methods after culture | cultivating in an osteoblast induction medium for 21 days. The result is shown in FIG. 3C. In FIG. 3C, a, b, and c are the control medium, d, e, and f are the osteoblast induction medium, and g, h, and i are the osteoblast induction medium + BMP-2 100 ng / ml. According to FIG. 3C, the number of cells positive for alkaline phosphatase activity staining, which is an indicator of osteoblast differentiation, increased, and the areas of von Kossa staining and alizarin red staining, which are indicators of calcification, were enlarged.
以上の評価試験１の結果から、脂肪組織由来再生細胞（ＡＤＲＣｓ）は、培養条件によって、種々の細胞へと分化する前駆細胞様の性状を備えることが確認できた。 From the results of the above evaluation test 1, it was confirmed that the adipose tissue-derived regenerative cells (ADRCs) have progenitor cell-like properties that differentiate into various cells depending on the culture conditions.
（評価試験２）
評価試験２では、実施例１の半月板再生材料１０を用いて動物実験を行い、半月板の再生程度について評価した。また、対照実験として、比較例１の材料２０を用いて、同様の動物実験を行った。以下、図４に沿って、評価試験２の手順を説明する。
(Evaluation test 2)
In the evaluation test 2, an animal experiment was performed using the meniscus regenerated material 10 of Example 1 to evaluate the degree of meniscus regeneration. As a control experiment, a similar animal experiment was conducted using the material 20 of Comparative Example 1. Hereinafter, the procedure of the evaluation test 2 will be described with reference to FIG.
［半月板損傷モデルの作成と半月板再生用材料の移植］
実験動物として複数のラットを用意した。ラットには全身麻酔を施し、十分に麻酔が効いた状態で剃毛後、膝前方皮膚を切開して関節まで達した後に、内側側副靭帯を頸骨付着部の顆間***の間で切離した。さらに周囲の軟組織は関節包から剥離して、内側半月板を十分に明示した状態で、図４に示すように、ラットの後脚の両膝において、その内側半月板２ａ，２ａ’の前側１／２を切除した。
[Creation of meniscus damage model and transplantation of meniscus regeneration material]
A plurality of rats were prepared as experimental animals. Rats were given general anesthesia, and after shaving in a fully anesthetized state, the skin on the front of the knee was incised to reach the joint, and then the medial collateral ligament was dissected between the intercondylar protuberances of the tibia attachment . Further, the surrounding soft tissue is detached from the joint capsule, and the inner meniscus is clearly shown. As shown in FIG. 4, the front side 1 of the inner meniscus 2a, 2a ′ is formed in both knees of the rear leg of the rat. / 2 was excised.
切除した半月板の部位に、ＡＤＲＣｓ溶液１３をアテロコラーゲンスポンジ１１に含浸させた実施例１の半月板再生用材料１０を、右膝の内側半月板２ａの切除部位に移植し、ナイロン糸で縫合して閉創した。対照として左膝の内側半月板２ａ’の切除部位に、アテロコラーゲンゲルのみ含ませた比較例１の材料２０を移植し、ナイロン糸で縫合して閉創した。 The meniscal regeneration material 10 of Example 1 in which the ADRCs solution 13 is impregnated with the atherocollagen sponge 11 in the excised meniscus site is transplanted to the excision site of the inner meniscus 2a of the right knee and sutured with nylon thread. Closed. As a control, the material 20 of Comparative Example 1 containing only atelocollagen gel was implanted at the excision site of the inner meniscus 2a 'of the left knee, and sutured with nylon thread to close the wound.
［免荷］
移植後１週間は、尾部懸垂によって、後脚の免荷を施した。
[Unloaded]
For one week after transplantation, the rear legs were unloaded by tail suspension.
［評価２−１］
移植して４週後と１２週後に、両膝の半月板２を摘出して観察した。図５Ａに、１２週後の半月板２の写真を示す。図５Ａの左図に示される実施例１の半月板再生用材料１０を移植した右膝の内側半月板２ａは、図５Ａの右図に示される比較例１の半月板再生用材料２０を移植した左膝の内側半月板２ａ’に比較して、内側半月板の前方半側の組織修復が亢進した。
[Evaluation 2-1]
At 4 weeks and 12 weeks after transplantation, the meniscus 2 of both knees was removed and observed. FIG. 5A shows a photograph of the meniscus 2 after 12 weeks. The medial meniscus 2a of the right knee transplanted with the meniscal regeneration material 10 of Example 1 shown in the left diagram of FIG. 5A is transplanted with the meniscal regeneration material 20 of Comparative Example 1 shown in the right diagram of FIG. 5A. Compared to the medial meniscus 2a ′ of the left knee, tissue repair on the anterior half of the medial meniscus was enhanced.
また、修復した組織を定量化するため、脛骨内顆関節面（図５Ｂの領域Ｂ）に対する半月板（図５Ｂの領域Ａ）の割合（領域Ａ／領域Ｂ）を算出した。図５Ｃは、算出した脛骨内顆関節面に対する半月板の割合を示すグラフである。この図５Ｃのグラフから解るように、実施例１の脂肪組織由来半月板再生用材料１０を移植したグループは、比較例１の半月板再生用材料２０を移植した対照グループに比べ、組織修復の割合が高いことが認められた（＊ｐ ＜ ０．０１）。 Further, in order to quantify the repaired tissue, the ratio (region A / region B) of the meniscus (region A in FIG. 5B) to the tibial condylar joint surface (region B in FIG. 5B) was calculated. FIG. 5C is a graph showing the ratio of the meniscus to the calculated tibial endocondylar joint surface. As can be seen from the graph of FIG. 5C, the group transplanted with the adipose tissue-derived meniscal regeneration material 10 of Example 1 was more repaired than the control group transplanted with the meniscal regeneration material 20 of Comparative Example 1. A high proportion was observed (* p <0.01).
［評価２−２］
移植して４週後と１２週後に、両膝の内側半月板２ａ，２ａ’を摘出し、Hematoxylin-Eosin（ＨＥ）（ヘマトキシリン−エオジン）染色、Toluidine Blue（トルイジンブルー）染色とII型コラーゲンの免疫組織染色からＡＤＲＣｓの再生能を評価した。
[Evaluation 2-2]
At 4 and 12 weeks after transplantation, the medial meniscus 2a and 2a ′ of both knees were removed, and hematoxylin-Eosin (HE) staining, Toluidine Blue staining and type II collagen The regeneration ability of ADRCs was evaluated from immunohistochemical staining.
ここでは、図６に移植後１２週の組織を組織学的に観察した結果を示す。図６（ａ）に示すように、ＡＤＲＣｓを含浸させたアテロコラーゲンスポンジ１１からなる実施例１の半月板再生用材料１０を移植した右側内側半月板２ａは、ヘマトキシリン−エオジン染色によれば、修復した前方欠損部に線維性軟骨とみられる組織修復が進んでいた。そして、既存の半月板（内側半月板２ａの後方半側）と同様に、軟骨の指標であるII型コラーゲンの発現と、酸性ムコ多糖を染めるトルイジンブルー染色陽性の領域が観察された。一方、図６（ｂ）に示すように、アテロコラーゲンスポンジ１１のみの比較例１の半月板再生用材料２０を移植した左側内側半月板２ａ’は、組織修復が認められなかった。 Here, FIG. 6 shows the result of histological observation of the tissue 12 weeks after transplantation. As shown in FIG. 6 (a), the right inner meniscus 2a transplanted with the meniscal regeneration material 10 of Example 1 made of atelocollagen sponge 11 impregnated with ADRCs was repaired according to hematoxylin-eosin staining. Tissue repair that seems to be fibrous cartilage was progressing in the anterior defect. Then, similar to the existing meniscus (the posterior half of the inner meniscus 2a), the expression of type II collagen, which is an indicator of cartilage, and the toluidine blue staining positive region for staining acid mucopolysaccharide were observed. On the other hand, as shown in FIG. 6B, the left inner meniscus 2a 'transplanted with the meniscal regeneration material 20 of Comparative Example 1 containing only the atelocollagen sponge 11 did not show tissue repair.
（評価試験３）
評価試験３では、全身の細胞が緑色蛍光タンパク質（ＧＦＰ）で標識されたＳＤ−Ｔｇ（ＣＡＧ−ＥＧＦＰ）グリーンラットのＡＤＲＣｓを用いて、実施例１と同様の半月板再生用材料１０を作製し、ヌードラットの右側の半月板切除部に移植して欠損部位への定着を評価した。また、対照として、アテロコラーゲンスポンジのみの比較例１の半月板再生用材料２０をヌードラットの左側の半月板切除部に移植した。また、修復された半月板の物性を評価するため、ナノインデンテーション法を用いて解析した。
(Evaluation Test 3)
In Evaluation Test 3, the same meniscus regeneration material 10 as in Example 1 was prepared using ADRCs of SD-Tg (CAG-EGFP) green rats in which whole body cells were labeled with green fluorescent protein (GFP). Then, transplantation was performed on the right meniscus excised part of the nude rat, and the establishment of the defect site was evaluated. Further, as a control, the meniscal regeneration material 20 of Comparative Example 1 containing only atelocollagen sponge was transplanted to the left meniscus resection portion of a nude rat. In addition, in order to evaluate the physical properties of the repaired meniscus, analysis was performed using the nanoindentation method.
［評価３−１］
図７（ａ）に、左右の脛骨内顆関節の明視野及び蛍光の写真を示し、図７（ｂ）に摘出した左右の半月板の明視野及び蛍光の写真を示す。また、図８に、II型コラーゲン、ＧＦＰの二重免疫組織染色（核染色）、及びこれらの重ね合わせの写真を示す。
[Evaluation 3-1]
FIG. 7 (a) shows bright field and fluorescent photographs of the left and right tibial condylar joints, and FIG. 7 (b) shows left and right meniscus bright field and fluorescent photographs. In addition, FIG. 8 shows photographs of type II collagen, GFP double immunohistochemical staining (nuclear staining), and their overlay.
移植したＡＤＲＣｓの修復後の分布を解析する目的で、緑色蛍光タンパク質（ＧＦＰ）で標識されたグリーンラット由来のＡＤＲＣｓを含む半月板再生用材料１０を移植すると、図７（ａ）、（ｂ）に実線で囲ったように、ＧＦＰ陽性細胞は移植相当部に局在した。 For the purpose of analyzing the post-repair distribution of transplanted ADRCs, when the meniscal regeneration material 10 containing green rat-derived ADRCs labeled with green fluorescent protein (GFP) is transplanted, FIG. 7 (a), (b) GFP-positive cells were localized in the transplantation-corresponding region as surrounded by a solid line.
さらに、図８に示すII型コラーゲンとＧＦＰの二重免疫組織染色の所見では、４週後ではII型コラーゲン陽性細胞は確認できなかったが、１２週後になるとＧＦＰ陽性の移植したＡＤＲＣｓの一部はII型コラーゲン陽性細胞であった。 Further, in the findings of double immunohistochemical staining of type II collagen and GFP shown in FIG. 8, type II collagen positive cells could not be confirmed after 4 weeks, but a part of GFP positive transplanted ADRCs was observed after 12 weeks. Were type II collagen positive cells.
以上の結果から、移植したＡＤＲＣｓは修復部位に定着して、さらに軟骨様細胞へ分化することが示唆された。 From the above results, it was suggested that the transplanted ADRCs settle at the repair site and further differentiate into cartilage-like cells.
［評価３−２］
また、膝関節に掛かる荷重を分散する能力を調べるために、修復された半月板の物性を評価した。ナノインデーテンション法による物性試験から、既存の半月板と比較して修復された半月板は僅かに弾性係数が高いが、近似した粘弾性を示した。
[Evaluation 3-2]
In addition, the physical properties of the repaired meniscus were evaluated to examine the ability to disperse the load on the knee joint. From the physical property test by the nanoindentation method, the repaired meniscus was slightly higher in elasticity than the existing meniscus, but showed an approximate viscoelasticity.
以上、評価試験１の結果から、実施例１に係るラット鼠径部の脂肪組織から採取したＡＤＲＣｓの一部は脂肪組織由来幹細胞を含み、培養条件によって、種々の細胞へと分化することが明らかになった。また、評価試験２、３の半月板損傷モデルの試験から、ＡＤＲＣｓによって健常組織に類似した半月板が誘導されること明らかとなった。このように、ＡＤＲＣｓは高い誘導能を有し、低侵襲で移植時に新鮮な細胞が十分量採取できることから、半月板再生医療の理想的な細胞ソースとして応用可能なことが示唆された。 As described above, it is clear from the results of Evaluation Test 1 that some ADRCs collected from the adipose tissue of the rat inguinal region according to Example 1 contain adipose tissue-derived stem cells and differentiate into various cells depending on the culture conditions. became. Moreover, it became clear from the test of the meniscus damage model of the evaluation tests 2 and 3 that the meniscus similar to a healthy tissue is induced | guided | derived by ADRCs. As described above, ADRCs have high inductive ability and are capable of collecting a sufficient amount of fresh cells at the time of transplantation with minimal invasiveness, suggesting that they can be applied as an ideal cell source for meniscal regenerative medicine.
以上、本発明の実施例を図面により詳述してきたが、上記各実施例は本発明の例示にしか過ぎないものであり、本発明は上記各実施例の構成にのみ限定されるものではない。本発明の要旨を逸脱しない範囲の設計の変更等があっても、本発明に含まれることは勿論である。 Although the embodiments of the present invention have been described in detail with reference to the drawings, each of the above embodiments is only an example of the present invention, and the present invention is not limited only to the configuration of each of the above embodiments. . Needless to say, changes in design and the like within the scope of the present invention are included in the present invention.
１０ 半月板再生用材料 １１ コラーゲンスポンジ
１２ 脂肪組織由来再生細胞（ＡＤＲＣｓ） １３ＡＤＲＣｓ溶液（混合溶液）
10 Meniscal regeneration material 11 Collagen sponge 12 Adipose tissue-derived regenerative cells (ADRCs) 13 ADRCs solution (mixed solution)
Claims (5)
コラーゲンスポンジと、
前記コラーゲンスポンジに含浸された脂肪組織由来再生細胞と、を有し、
前記脂肪組織由来再生細胞は、生体内から採取された脂肪組織から分離され培養されることなく得られた、脂肪組織由来幹細胞を含む細胞集団であることを特徴とする半月板再生用材料。 A material for regenerating meniscus used to regenerate at least a part of the meniscus of a knee joint, which is used to be implanted in the knee joint,
Collagen sponge,
Have a, and fat tissue derived regenerative cells impregnated before Symbol collagen sponge,
The material for regenerating meniscus, wherein the adipose tissue-derived regenerative cells are a cell population containing adipose tissue-derived stem cells obtained without being separated and cultured from adipose tissue collected from the living body .
生体内から脂肪組織を採取する工程と、
採取された前記脂肪組織から脂肪組織由来幹細胞を含む細胞集団である脂肪組織由来再生細胞を分離する工程と、
分離した前記脂肪組織由来再生細胞を、培養することなくコラーゲンスポンジに含浸させる工程と、を有することを特徴とする半月板再生用材料の作製方法。 In order to regenerate at least part of the meniscus of the knee joint, a method for producing a meniscal regeneration material used to be implanted in the knee joint,
Collecting adipose tissue from the living body ;
And separating the adipose tissue derived regenerative cells from harvested the adipose tissue is a cell population containing adipose tissue-derived stem cells,
And a step of impregnating collagen sponge with the separated adipose tissue-derived regenerative cells without culturing. A method for producing a meniscal regeneration material, comprising:
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| JP2007508018A (en) * | 2003-10-08 | 2007-04-05 | べト−ステム インコーポレイテッド | Method for preparing and using a novel stem cell composition, and kit containing the composition |
| JP2010075201A (en) * | 2001-03-23 | 2010-04-08 | Histogenics Corp | Tissue construct and multi-layered tissue construct |
| JP2014183896A (en) * | 2013-03-22 | 2014-10-02 | Osaka Medical College | Semilunar plate regeneration backing material |
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| JP2007508018A (en) * | 2003-10-08 | 2007-04-05 | べト−ステム インコーポレイテッド | Method for preparing and using a novel stem cell composition, and kit containing the composition |
| JP2014183896A (en) * | 2013-03-22 | 2014-10-02 | Osaka Medical College | Semilunar plate regeneration backing material |
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| 日本整形外科学会雑誌, vol. 87, no. 8, JPN6017032557, 2013, pages P.S1403 * |
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