JP2024002617A - Meniscus regeneration promoter, materials for meniscus regeneration, and method for making materials for meniscus regeneration - Google Patents
Meniscus regeneration promoter, materials for meniscus regeneration, and method for making materials for meniscus regeneration Download PDFInfo
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Abstract
Description
新規性喪失の例外適用申請有り There is an application for exception to loss of novelty.
本発明は、半月板再生促進剤、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 meniscal regeneration promoter, a meniscal regeneration material, and a method for producing a meniscal regeneration material.
膝半月板は、荷重に対する力学分散としての役割を担う一方で、生物学的治癒能力の低さから、一旦受傷すると修復が困難である。そのため、半月板はいったん損傷されるとＡＤＬ（Ａｃｔｉｖｉｔｉｅｓ ｏｆ Ｄａｉｌｙ Ｌｉｖｉｎｇ：日常生活動作）の低下をもたらす。
半月板修復には血流の関与が重要であると言われており、血行野（関節包側）での損傷は治癒する可能性が高いとされている。しかし、無血行野での損傷は半月板の切除を余儀なくされており、縫合後の再手術率も２０％以上と少なくない（非特許文献１）。
While the knee meniscus plays a role in mechanically distributing the load, it is difficult to repair once injured due to its low biological healing ability. Therefore, once the meniscus is damaged, it causes a decline in ADL (Activities of Daily Living).
It is said that the involvement of blood flow is important for meniscus repair, and damage in the blood circulation field (on the side of the joint capsule) is said to have a high possibility of healing. However, damage in the avascular field necessitates removal of the meniscus, and the reoperation rate after suturing is not low, at over 20% (Non-Patent Document 1).
縫合術時に付加的処置として骨髄刺激、ラスピングがあるが、その効果は限定的である。海外では人工半月板が認められているが、日本では認められておらず、半月板の再建手術は難しい。
半月板切除後欠損に対する手術としては、自家組織（半腱様筋腱）を用いた半月板再建術が行われている。しかし、治療成績を向上させるため、自家腱の軟骨化を促す追加処置が求められている。
Bone marrow stimulation and rasping are available as additional treatments during suturing, but their effects are limited. Artificial menisci are allowed overseas, but not in Japan, making meniscal reconstruction surgery difficult.
Meniscal reconstruction using autologous tissue (semitendinosus tendon) is performed as surgery for defects after meniscectomy. However, in order to improve treatment results, additional treatments that promote cartilage of autologous tendons are required.
本発明は、半腱様筋腱組織を用いた半月板再建術において有用な半月板再生促進剤を提供することを目的のひとつとする。 One of the objects of the present invention is to provide a meniscal regeneration promoter useful in meniscal reconstruction using semitendinosus tendon tissue.
本発明者らは、上記課題を解決すべく鋭意検討を重ねた結果、半腱様筋腱組織に副甲状腺ホルモン（ＰＴＨ）を注入することで、半月板組織を再生できることを知得し、本発明を完成させた。 As a result of intensive studies to solve the above problems, the present inventors discovered that meniscal tissue can be regenerated by injecting parathyroid hormone (PTH) into the semitendinosus tendon tissue. Completed the invention.
［１］ 副甲状腺ホルモン及びそのＮ末端フラグメントからなる群から選択される少なくとも１種を有効成分とする半月板再生促進剤。
［２］ ヒト副甲状腺ホルモン又はそのＮ末端から３２～３６番目までのアミノ酸残基からなるポリペプチドを有効成分とする、［１］に記載の半月板再生促進剤。
［３］ 配列番号１（SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF）で表されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列からなるポリペプチドを有効成分とする半月板再生促進剤。
［４］ 配列番号２（SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ）で表されるアミノ酸配列のＮ末端側１～３４番目のアミノ酸配列と９０％以上の配列同一性を有し、かつ前記配列番号２で表されるアミノ酸配列のＮ末側１～８４番目のアミノ酸配列と７０％以上の配列同一性を有するアミノ酸配列からなるポリペプチドを有効成分とする半月板再生促進剤。
［５］ 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するように用いられる半月板再生用材料であって、
提供者から提供された半腱様筋腱組織と、
前記半腱様筋腱組織に注入された［１］～［４］のいずれかに記載の半月板再生促進剤と、
を含む、半月板再生用材料。
［６］ 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するように用いられる半月板再生用材料の作製方法であって、
提供者から提供された半腱様筋腱組織に［１］～［４］のいずれかに記載の半月板再生促進剤を生体外で注入する工程と、
を含む半月板再生用材料の作製方法。
［７］ 半腱様筋腱組織を用いる半月板再建方法において、前記半腱様筋腱組織と［１］～［４］のいずれかに記載の半月板再生促進剤とを接触させる、半月板再建方法。
［８］ 前記半腱様筋腱組織を提供者から採取した後、かつ前記半腱様筋腱組織を移植者に移植する前に、前記半腱様筋腱組織と前記半月板再生促進剤とを接触させる、［７］に記載の半月板再建方法。
［９］ 前記半腱様筋腱組織を提供者から採取する前に、前記半腱様筋腱組織と前記半月板再生促進剤とを接触させる、［７］に記載の半月板再建方法。
［１０］ 前記半腱様筋腱組織がアキレス腱組織である、［７］～［９］のいずれかに記載の半月板再建方法。
［１１］ 前記半腱様筋腱組織の提供者及び移植者が同一である、［７］～［１０］のいずれかに記載の半月板再建方法。
［１２］ 前記半腱様筋腱組織の提供者及び移植者がいずれもヒトである、［７］～［１１］のいずれかに記載の半月板再建方法。
[1] A meniscal regeneration promoter containing at least one member selected from the group consisting of parathyroid hormone and its N-terminal fragment as an active ingredient.
[2] The meniscal regeneration promoter according to [1], which contains human parathyroid hormone or a polypeptide consisting of the 32nd to 36th amino acid residues from its N-terminus as an active ingredient.
[3] A meniscal regeneration promoter containing as an active ingredient a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF).
[4] Having 90% or more sequence identity with the 1st to 34th amino acid sequence on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ), and A meniscal regeneration promoter containing as an active ingredient a polypeptide consisting of an amino acid sequence having 70% or more sequence identity with the 1st to 84th amino acid sequence on the N-terminal side.
[5] A meniscal regeneration material used to be transplanted into the knee joint in order to regenerate at least a part of the meniscus of the knee joint,
semitendinosus tendon tissue provided by a donor;
The meniscal regeneration promoter according to any one of [1] to [4] injected into the semitendinosus tendon tissue;
Materials for meniscal regeneration, including:
[6] A method for producing a meniscus regeneration material used to be transplanted into the knee joint in order to regenerate at least a part of the meniscus of the knee joint, comprising:
Injecting the meniscal regeneration promoter according to any one of [1] to [4] into the semitendinosus tendon tissue provided by the donor in vitro;
A method for producing a material for meniscal regeneration, including:
[7] A meniscal reconstruction method using semitendinosus tendon tissue, which comprises bringing the semitendinosus tendon tissue into contact with the meniscal regeneration promoter according to any one of [1] to [4]. Reconstruction method.
[8] After the semitendinosus tendon tissue is collected from the donor and before the semitendinosus tendon tissue is transplanted to the recipient, the semitendinosus tendon tissue and the meniscal regeneration promoter are combined. The meniscal reconstruction method according to [7], which comprises contacting the
[9] The meniscal reconstruction method according to [7], wherein the semitendinosus tendon tissue is brought into contact with the meniscal regeneration promoter before the semitendinosus tendon tissue is collected from the donor.
[10] The meniscal reconstruction method according to any one of [7] to [9], wherein the semitendinosus tendon tissue is Achilles tendon tissue.
[11] The meniscal reconstruction method according to any one of [7] to [10], wherein the donor and transplanter of the semitendinosus tendon tissue are the same.
[12] The meniscal reconstruction method according to any one of [7] to [11], wherein both the donor and transplanter of the semitendinosus tendon tissue are humans.
本発明は、半腱様筋腱組織を用いた半月板再建術において有用な半月板再生促進剤を提供する。本発明は。また、半月板再生用材料及び半月板再生用材料の作製方法を提供する。 The present invention provides a meniscal regeneration promoter useful in meniscal reconstruction using semitendinosus tendon tissue. The present invention is. The present invention also provides a material for regenerating a meniscus and a method for producing the material for regenerating a meniscus.
以下、本発明の半月板再生促進剤、半月板再生用材料及び半月板再生用材料の作製方法について、詳細に説明する。 Hereinafter, the meniscal regeneration promoter, the meniscal regeneration material, and the method for producing the meniscal regeneration material of the present invention will be described in detail.
本発明において、「～」又は「－」を用いて数値範囲を表す場合、その両側の数値をその数値範囲内に含むものとする。 In the present invention, when a numerical range is expressed using "~" or "-", the numerical values on both sides are included within the numerical range.
［半月板再生促進剤］
本発明の半月板再生促進剤の一実施形態は、副甲状腺ホルモン及びそのＮ末端フラグメントからなる群から選択される少なくとも１種を有効成分とする半月板再生促進剤である。
[Meniscal regeneration promoter]
One embodiment of the meniscal regeneration promoter of the present invention is a meniscal regeneration promoter containing at least one member selected from the group consisting of parathyroid hormone and its N-terminal fragment as an active ingredient.
副甲状腺とは、甲状腺に隣接して存在する内分泌腺の１種である。動物のなかでも四足動物である哺乳類、鳥類、爬虫類及び両生類には存在するが、魚類には存在しない。ヒトでは副甲状腺は２対計４個が存在するが、動物種によっては３対や４対有することもある。
副甲状腺は、血液中のカルシウムイオン濃度を一定に保つための内分泌器官である。細胞外（血液中）のカルシウムは、筋肉の収縮、神経刺激の伝達、血液の凝固、ホルモンの分泌など、多くの重要な生理機能を調節している。特に細胞外から細胞内へのカルシウムの流入は、多くの現象のシグナルとして働くので、血液中のカルシウム濃度は一定に保たれていなければならない。四足動物はカルシウムを食餌からしか得ることができないので、骨にカルシウムを蓄積し、濃度が低下する度に血液中に適量溶かし出す必要がある。副甲状腺は、血液中のカルシウム濃度を監視し、副甲状腺ホルモンを血液中に分泌して全身の骨からカルシウムを溶かし出し、血液中のカルシウム濃度を一定に機能を担っている。
The parathyroid gland is a type of endocrine gland that exists adjacent to the thyroid gland. Among animals, it exists in mammals, birds, reptiles, and amphibians that are quadrupeds, but it does not exist in fish. In humans, there are two pairs of parathyroid glands, four in total, but some animal species may have three or four pairs.
The parathyroid glands are endocrine organs that maintain a constant concentration of calcium ions in the blood. Extracellular (blood) calcium regulates many important physiological functions, including muscle contraction, transmission of nerve impulses, blood clotting, and hormone secretion. In particular, the influx of calcium from the outside of the cell into the cell serves as a signal for many phenomena, so the concentration of calcium in the blood must be kept constant. Quadrupeds can only obtain calcium from their diet, so they must accumulate calcium in their bones and dissolve it into their blood whenever the concentration drops. The parathyroid glands monitor the calcium concentration in the blood, secrete parathyroid hormone into the blood, dissolve calcium from bones throughout the body, and maintain a constant calcium concentration in the blood.
副甲状腺ホルモンは副甲状腺から分泌されるペプチドホルモンであり、パラトルモン（ｐａｒａｔｈｏｒｍｏｎｅ，ＰＴＨ）とも呼ばれる。ＰＴＨは、血液のカルシウムの濃度を増加させるように働き、逆に甲状腺から分泌されるカルシトニンはカルシウムを減少させるように働く。ＰＴＨは、血中のカルシウム濃度を増加させるが、パラトルモン受容体（ＰＴＨ受容体）は骨、腸、腎臓の３箇所の臓器に発現が見られる。
ヒト副甲状腺ホルモンは８４アミノ酸残基からなるポリペプチド（配列番号２）であり、ＰＴＨ（１－８４）とも表記される。ヒトＰＴＨのＮ末端３４アミノ酸残基からなるポリペプチド（ＰＴＨ（１－３４）；配列番号１）は、骨粗鬆症治療薬テリパラチドとして臨床応用されている。上述のとおりＰＴＨには骨吸収促進作用があり、副甲状腺機能亢進症で見られるように持続的なＰＴＨ投与は骨密度を低下させるが、間欠的投与では逆に骨形成を促進し、骨折リスクを低下させることが知られている。
副甲状腺ホルモンとしては、哺乳類、鳥類、爬虫類又は両生類のいずれの副甲状腺ホルモンでもよいが、哺乳類の副甲状腺ホルモンが好ましく、霊長類の副甲状腺ホルモンがより好ましく、ヒトの副甲状腺ホルモンがさらに好ましい。
Parathyroid hormone is a peptide hormone secreted from the parathyroid glands, and is also called parathormone (PTH). PTH works to increase the concentration of calcium in the blood, and conversely, calcitonin secreted from the thyroid gland works to decrease calcium. PTH increases blood calcium concentration, and parathormone receptor (PTH receptor) is expressed in three organs: bones, intestines, and kidneys.
Human parathyroid hormone is a polypeptide (SEQ ID NO: 2) consisting of 84 amino acid residues, and is also written as PTH (1-84). A polypeptide consisting of the N-terminal 34 amino acid residues of human PTH (PTH(1-34); SEQ ID NO: 1) has been clinically applied as the osteoporosis therapeutic drug teriparatide. As mentioned above, PTH has the effect of promoting bone resorption, and continuous administration of PTH decreases bone density, as seen in hyperparathyroidism, but intermittent administration promotes bone formation and increases the risk of fracture. is known to reduce
The parathyroid hormone may be any mammalian, avian, reptile, or amphibian parathyroid hormone, but mammalian parathyroid hormone is preferred, primate parathyroid hormone is more preferred, and human parathyroid hormone is even more preferred.
副甲状腺ホルモンのＮ末端フラグメントは、上述した副甲状腺ホルモンのＮ末端を含むフラグメントであれば特に限定されないが、副甲状腺ホルモンのＮ末端から３２～３６番目までのアミノ酸残基からなるポリペプチドが好ましく、ヒト副甲状腺ホルモン（ＰＴＨ（１－８４））のＮ末端から３２～３６番目までのアミノ酸残基からなるポリペプチドがより好ましく、ヒト副甲状腺ホルモン（ＰＴＨ（１－８４））のＮ末端から３４アミノ酸残基のポリペプチド（ＰＴＨ（１－３４））がさらに好ましい。
ＰＴＨ（１－３４）としては、テリパラチド（遺伝子組換え）（一般名）、フォルテオ（商品名、イーライ リリー社製、登録商標）、テリボン（商品名、旭化成ファーマ社製、登録商標））など市販品を使用してもよい。
The N-terminal fragment of parathyroid hormone is not particularly limited as long as it is a fragment containing the N-terminus of parathyroid hormone, but preferably a polypeptide consisting of amino acid residues from the 32nd to 36th amino acid residues from the N-terminus of parathyroid hormone. , a polypeptide consisting of amino acid residues from the 32nd to 36th amino acid residues from the N-terminus of human parathyroid hormone (PTH(1-84)) is more preferable; More preferred is a polypeptide of 34 amino acid residues (PTH(1-34)).
PTH (1-34) is commercially available, such as teriparatide (genetically recombinant) (generic name), Forteo (trade name, manufactured by Eli Lilly, registered trademark), and Teribon (trade name, manufactured by Asahi Kasei Pharma, registered trademark). You may use products.
本発明の半月板再生促進剤の別の一実施形態は、配列番号１（SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF）で表されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列からなるポリペプチドを有効成分とする半月板再生促進剤である。
配列番号１で表されるアミノ酸配列は、ＰＴＨ（１－３４）のアミノ酸配列である。
配列番号１のアミノ酸配列とあるアミノ酸配列との配列同一性は、配列番号１のアミノ酸配列と前記あるアミノ酸配列とでペアワイズにアラインメントを行い、比較可能な全ポジションについて、一致（マッチ）及び不一致（アンマッチ）を判定し、比較可能な全ポジション中の一致ポジションの割合（百分率）を配列同一性とする。対応するポジションが無い場合はギャップを挿入して長さを揃えるが、配列番号１のアミノ酸配列のＮ末端よりも左側及びＣ末端よりも右側は比較可能な全ポジションにカウントしない。
Another embodiment of the meniscal regeneration promoter of the present invention is a meniscal whose active ingredient is a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF). It is a board regeneration accelerator.
The amino acid sequence represented by SEQ ID NO: 1 is that of PTH (1-34).
Sequence identity between the amino acid sequence of SEQ ID NO: 1 and a certain amino acid sequence is determined by performing pairwise alignment between the amino acid sequence of SEQ ID NO: 1 and the above-mentioned amino acid sequence, and determining whether there are matches or mismatches for all positions that can be compared. The sequence identity is defined as the percentage of matching positions among all comparable positions. If there is no corresponding position, a gap is inserted to make the lengths uniform, but the positions to the left of the N-terminus and to the right of the C-terminus of the amino acid sequence of SEQ ID NO: 1 are not counted as all comparable positions.
本発明の半月板再生促進剤のまた別の一実施形態は、配列番号２（SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ）で表されるアミノ酸配列のＮ末端側１～３４番目のアミノ酸配列と９０％以上の配列同一性を有し、かつ前記配列番号２で表されるアミノ酸配列のＮ末側１～８４番目のアミノ酸配列と７０％以上の配列同一性を有するアミノ酸配列からなるポリペプチドを有効成分とする半月板再生促進剤である。
配列番号１で表されるアミノ酸配列は、ＰＴＨ（１－８４）のアミノ酸配列である。
配列同一性の計算方法は上述したとおりである。
Another embodiment of the meniscal regeneration promoter of the present invention has 90% or more sequence identity with the 1st to 34th amino acid sequence on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ). A meniscal regeneration promoter containing as an active ingredient a polypeptide comprising an amino acid sequence having 70% or more sequence identity with the 1st to 84th amino acid sequence on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2. It is.
The amino acid sequence represented by SEQ ID NO: 1 is that of PTH (1-84).
The method for calculating sequence identity is as described above.
上述した有効成分は、その製薬学上許容しうる塩又はエステルであってもよい。例えば、グルタミン酸残基（Ｅ）又はアスパラギン酸残基（Ｄ）の側鎖カルボキシ基は、塩又はエステルの形態であってもよい。また、例えば、リシン（Ｋ），アルギニン（Ｒ）、ヒスチジン（Ｈ）又はトリプトファン（Ｗ）の側鎖アミノ基（イミノ基）は、塩の形態であってもよい。 The above-mentioned active ingredients may be their pharmaceutically acceptable salts or esters. For example, the side chain carboxy group of the glutamic acid residue (E) or the aspartic acid residue (D) may be in the form of a salt or an ester. Further, for example, the side chain amino group (imino group) of lysine (K), arginine (R), histidine (H), or tryptophan (W) may be in the form of a salt.
本発明の半月板再生促進剤は、上述した有効成分の他に、水及び製剤添加剤を含んでもよい。前記製剤添加剤としては、例えば、賦形剤、結合剤、崩壊剤及び滑沢剤が挙げられるがこれらに限定されない。 The meniscal regeneration promoter of the present invention may contain water and formulation additives in addition to the above-mentioned active ingredients. Examples of the formulation additives include, but are not limited to, excipients, binders, disintegrants, and lubricants.
本発明の半月板再生促進剤の投与方法は、特に限定されないが、一期的で簡便な方法であることが望まれることから、半腱様筋腱組織を用いる半月板再建方法において、前記半腱様筋腱組織を提供者から採取した後、かつ前記半腱様筋腱組織を移植者に移植する前に、前記半腱様筋腱組織と本発明の半月板再生促進剤とを接触させること、又は、前記半腱様筋腱組織を提供者から採取する前に、前記半腱様筋腱組織と本発明の半月板再生促進剤とを接触させること、が好ましい。前記半腱様筋腱組織と本発明の半月板再生促進剤とを接触させる方法としては、例えば、本発明の半月板再生促進剤を前記半腱様筋腱組織に注入する方法が挙げられる。
本発明の半月板再生促進剤を前記半腱様筋腱組織に注入する場合の注入量は、特に限定されないが、前記半腱様筋腱組織の１０ｇあたり、本発明の半月板再生促進剤の有効成分量として、１０～１２０μｇが好ましく、２５～１００μｇがより好ましく、５０～７０μｇがさらに好ましい。
The method for administering the meniscal regeneration promoting agent of the present invention is not particularly limited, but since it is desired to be a one-time and simple method, in the meniscal reconstruction method using semitendinosus tendon tissue, After harvesting the tendinoid muscle tendon tissue from the donor and before transplanting the semitendinosus tendon tissue to the recipient, the semitendinosus tendon tissue is brought into contact with the meniscal regeneration promoter of the present invention. Alternatively, it is preferable that the semitendinosus tendon tissue is brought into contact with the meniscal regeneration promoter of the present invention before the semitendinosus tendon tissue is collected from the donor. Examples of the method of bringing the semitendinosus tendon tissue into contact with the meniscal regeneration promoter of the present invention include a method of injecting the meniscal regeneration promoter of the present invention into the semitendinosus tendon tissue.
When injecting the meniscal regeneration promoter of the present invention into the semitendinosus tendon tissue, the injection amount is not particularly limited, but the amount of the meniscal regeneration promoter of the present invention per 10 g of the semitendinosus tendon tissue is The amount of active ingredient is preferably 10 to 120 μg, more preferably 25 to 100 μg, even more preferably 50 to 70 μg.
本発明の半月板再生促進剤を用いる対象は、副甲状腺及び半月板を有する動物であれば特に限定されないが、ヒト又は非ヒト哺乳動物が好ましく、ヒトがより好ましい。非ヒト哺乳動物としては、ゴリラ，サル、チンパンジー、イヌ、ネコ、ウシ、ウマ、ブタ等が挙げられるが、これらに限定されない。 The subject to whom the meniscal regeneration promoter of the present invention is used is not particularly limited as long as it is an animal having parathyroid glands and a meniscus, but humans or non-human mammals are preferable, and humans are more preferable. Non-human mammals include, but are not limited to, gorillas, monkeys, chimpanzees, dogs, cats, cows, horses, pigs, and the like.
［半月板再生用材料及び半月板再生用材料の作製方法］
本発明の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するように用いられる半月板再生用材料であって、提供者から提供された半腱様筋腱組織と、前記半腱様筋腱組織に注入された本発明の半月板再生促進剤と、を含む、半月板再生用材料である。
前記半腱様筋腱組織としては、骨格筋である半腱様筋（紡錘筋）が骨に付着する部分の筋肉主体部にある結合組織であれば特に限定されないが、アキレス腱が好ましい。なお、半腱様筋腱組織を採取した後、腱は再生し、再生腱はほぼ正常な機能を果たすと考えられている。
[Meniscal regeneration material and method for producing meniscal regeneration material]
The meniscal regeneration material of the present invention is a meniscal regeneration material used to be transplanted into the knee joint in order to regenerate at least a part of the meniscus of the knee joint, and is provided by a donor. This is a material for meniscus regeneration, comprising a semitendinosus tendon tissue and a meniscal regeneration promoter of the present invention injected into the semitendinosus tendon tissue.
The semitendinosus tendon tissue is not particularly limited as long as it is connective tissue located in the main body of the muscle where the skeletal muscle semitendinosus (fusiform muscle) attaches to the bone, but Achilles tendon is preferred. It is believed that after the semitendinosus tendon tissue is harvested, the tendon regenerates and the regenerated tendon functions almost normally.
本発明の半月板再生用材料の作製方法は、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するように用いられる半月板再生用材料の作製方法であって、提供者から提供された半腱様筋腱組織に本発明の半月板再生促進剤を生体外で注入する工程と、を含む半月板再生用材料の作製方法である。
半腱様筋腱組織に本発明の半月板再生促進剤を生体外で注入する方法は、特に限定されず、例えば、注射器等を用いて注入することができる。
本発明の半月板再生促進剤の注入量は、特に限定されないが、前記半腱様筋腱組織の１０ｇあたり、本発明の半月板再生促進剤の有効成分量として、１０～１２０μｇが好ましく、２５～１００μｇがより好ましく、５０～７０μｇがさらに好ましい。
The method for producing a material for meniscal regeneration of the present invention is a method for producing a material for meniscal regeneration that is used to be transplanted into the knee joint in order to regenerate at least a part of the meniscus of the knee joint, comprising: This is a method for producing a material for meniscal regeneration, which includes the step of injecting the meniscal regeneration promoter of the present invention into semitendinosus tendon tissue provided by a donor in vitro.
The method of injecting the meniscal regeneration promoter of the present invention into the semitendinosus tendon tissue outside the body is not particularly limited, and can be injected using, for example, a syringe.
The injection amount of the meniscal regeneration promoter of the present invention is not particularly limited, but the amount of active ingredient of the meniscal regeneration promoter of the present invention per 10 g of the semitendinosus tendon tissue is preferably 10 to 120 μg, and 25 ~100 μg is more preferred, and 50-70 μg is even more preferred.
［半月板再建方法］
本発明の半月板再建方法は、半腱様筋腱組織を用いる半月板再建方法において、前記半腱様筋腱組織と本発明の半月板再生促進剤とを接触させる、半月板再建方法である。
半腱様筋腱組織は上述したとおりである。
半腱様筋腱組織と本発明の半月板再生促進剤とを接触させる方法としては、本発明の半月板再生促進剤を前記半腱様筋腱組織に注入する方法が挙げられる。この場合の本発明の半月板再生促進剤の注入量は、特に限定されないが、前記半腱様筋腱組織の１０ｇあたり、本発明の半月板再生促進剤の有効成分量として、１０～１２０μｇが好ましく、２５～１００μｇがより好ましく、５０～７０μｇがさらに好ましい。
[Meniscal reconstruction method]
The meniscal reconstruction method of the present invention is a meniscal reconstruction method using semitendinosus tendon tissue, which comprises bringing the semitendinosus tendon tissue into contact with the meniscal regeneration promoter of the present invention. .
The semitendinosus tendon tissue is as described above.
Examples of the method of bringing the semitendinosus tendon tissue into contact with the meniscal regeneration promoter of the present invention include a method of injecting the meniscal regeneration promoter of the present invention into the semitendinosus tendon tissue. In this case, the injection amount of the meniscal regeneration promoter of the present invention is not particularly limited, but the amount of active ingredient of the meniscal regeneration promoter of the present invention per 10 g of the semitendinosus tendon tissue is 10 to 120 μg. It is preferably 25 to 100 μg, more preferably 50 to 70 μg.
半腱様筋腱組織と本発明の半月板再生促進剤とを接触させるタイミングは、前記半腱様筋腱組織を提供者から採取した後、かつ前記半腱様筋腱組織を移植者に移植する前、又は、前記半腱様筋腱組織を提供者から採取する前、が好ましい。 The timing of contacting the semitendinosus tendon tissue with the meniscal regeneration promoter of the present invention is after the semitendinosus tendon tissue is harvested from the donor and after the semitendinosus tendon tissue is transplanted to the recipient. or before the semitendinosus tendon tissue is harvested from the donor.
前記半腱様筋腱組織の提供者及び前記半腱様筋腱組織の移植者は、同一個体であってもよいし、異なる個体であってもよい。前記提供者及び前記移植者はいずれもヒトであることが好ましい。 The donor of the semitendinosus tendon tissue and the recipient of the semitendinosus tendon tissue may be the same individual or different individuals. Preferably, both the donor and the recipient are humans.
以下では実施例によって本発明をより具体的に説明するが、本発明は後述する実施例によって限定されるものではない。 EXAMPLES The present invention will be explained in more detail with reference to examples below, but the present invention is not limited to the examples described below.
以下の実施例では、腱組織に副甲状腺ホルモン（ＰＴＨ（１－３４））を注入し、半月板切除部位へ移植することで、正常半月板に近い軟骨様組織を再現した。 In the following example, parathyroid hormone (PTH(1-34)) was injected into tendon tissue and transplanted to the meniscectomy site, thereby reproducing cartilage-like tissue similar to a normal meniscus.
［実施例１］
野生型Ｌｅｗｉｓ ラット（８～１０週齢、オス）１２匹よりアキレス腱を採取、ＰＢＳ ｗａｓｈ後１ｍｍ角にカットしコラゲナーゼ処理を行い、培地（Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ ＋ Ｆｅｔａｌ ｂｏｖｉｎｅ ｓｅｒｕｍ ＋ ｐｅｎｉｃｉｌｌｉｎ／ｓｔｒｅｐｔｏｍｙｃｉｎ）で培養、接着した細胞を腱細胞として用いた。
１回継代ののちに１２ｗｅｌｌプレートに１×１０５／ｗｅｌｌの濃度で播種し、ＰＴＨ（１－３４）を（テリボン、旭化成ファーマ社製）０、１、１０、１００ｎＭの濃度で４群に分けて添加処理後、２８日でアルシアンブルー染色を行ったところ、１０ｎＭで最も染色性がよく、軟骨基質産生が見られた（図１）。
ＰＴＨ（１－３４）を１０ｎＭ／ｍｌ添加した腱細胞を１４日、２８日で回収し、ＰＴ－ＰＣＲを行ったところ、軟骨基質を示すＣｏｌ２、Ｓｏｘ９、Ｒｕｎｘ２遺伝子の発現増強がみられ、ＰＴＨレセプターの発現増加もみられた（図２、図３）。
[Example 1]
Achilles tendons were collected from 12 wild-type Lewis rats (8-10 weeks old, male), washed with PBS, cut into 1 mm squares, treated with collagenase, and mixed with medium (Dulbecco's Modified Eagle Medium + Fetal bovine serum + penicillin). /streptomycin) The cells that were cultured and adhered were used as tendon cells.
After the first passage, the cells were seeded in a 12-well plate at a concentration of 1 x 10 5 /well, and PTH (1-34) (Teribon, manufactured by Asahi Kasei Pharma) was added to 4 groups at concentrations of 0, 1, 10, and 100 nM. When Alcian blue staining was performed 28 days after the addition treatment, staining was best at 10 nM, and cartilage matrix production was observed (FIG. 1).
Tendon cells to which 10 nM/ml of PTH (1-34) had been added were collected on the 14th and 28th day, and PT-PCR was performed. Increased receptor expression was also observed (Figures 2 and 3).
［実施例２］
アキレス腱へのＰＴＨ（１－３４）注入（ｉｎ ｖｉｖｏ実験）
ラットアキレス腱部にＰＴＨ（１－３４）（テリボン、旭化成ファーマ社製）を０μｇ、９、２２．５、４５ｍｇ／１５μＬ注入し、術後４週、８週で犠牲死させ、腱組織を評価した。
ＰＴＨを注入したアキレス腱組織はいずれの濃度においても術後４週よりトルイジンブルー染色にて異染性を認めたが、２２．５ｍｇ以上では術後８週の組織で一部骨化がみられた（図４）。
９μｇ／１５μＬの投与量で半月板が再生された。
[Example 2]
PTH (1-34) injection into Achilles tendon (in vivo experiment)
0 μg, 9, 22.5, 45 mg/15 μL of PTH (1-34) (Teribon, manufactured by Asahi Kasei Pharma) was injected into the Achilles tendon of rats, and the rats were sacrificed at 4 and 8 weeks after surgery, and the tendon tissue was evaluated. .
The Achilles tendon tissue injected with PTH showed metachromaticity in toluidine blue staining from 4 weeks after surgery at any concentration, but some ossification was observed in the tissue 8 weeks after surgery at doses of 22.5 mg or higher. (Figure 4).
The meniscus was regenerated at a dose of 9 μg/15 μL.
［実施例３］
ＰＴＨ（１－３４）注入アキレス腱を用いた半月板再建（ｉｎ ｖｉｖｏ実験）
ラットの内側半月板部分欠損モデルを作成し、ＰＴＨ（１－３４）（テリボン、旭化成ファーマ社製）を９μｇ／１５μＬ注入したアキレス腱を採取し半月板欠損部に縫合することで半月板再建モデルとした。対称群としてＰＢＳを１５μＬ注入したアキレス腱による再建モデルを作成した。４週、８週で犠牲死させ組織を評価した。
ＰＴＨ注入腱を用いた半月板再建モデルでは対称群と比較してｍｅｎｉｓｃｕｓ ｃｏｖｅｒ ｒａｔｉｏが高く、脛骨関節面Ｉｎｄｉａ ｉｎｋ ｓｔａｉｎでの染色率が低かった。半月板組織のトルイジンブルー染色ではＰＴＨ注入腱で異染性を認め、ｍｏｄｉｆｉｅｄ Ｐａｕｌｉ ｓｃｏｒｅで４週、８週共にＰＴＨ群が優位に高い結果となった。脛骨関節面組織のｍｏｄｉｆｉｅｄ Ｗａｋｉｔａｎｉ ｓｃｏｒｅでは術後８週においてＰＴＨ群のほうが有位に高かった。
マクロ評価の結果を図５、半月板組織評価の結果を図６に、脛骨組織評価の結果を図７に示す。
図５～図７から、より正常半月板に近い組織形態と、高い半月板被覆率、軟骨保護作用が認められた。
[Example 3]
Meniscal reconstruction using PTH(1-34)-injected Achilles tendon (in vivo experiment)
A partial medial meniscal defect model was created in rats, and the Achilles tendon injected with 9 μg/15 μL of PTH (1-34) (Teribon, manufactured by Asahi Kasei Pharma) was harvested and sutured to the meniscus defect to create a meniscal reconstruction model. did. As a control group, a reconstruction model using the Achilles tendon was created in which 15 μL of PBS was injected. The sacrificed tissues were evaluated at 4 and 8 weeks.
In the meniscal reconstruction model using PTH-injected tendon, the meniscus cover ratio was higher and the staining rate of the tibial articular surface India ink stain was lower than in the symmetric group. Toluidine blue staining of the meniscal tissue showed metachromatism in the PTH-injected tendon, and the modified Pauli score was significantly higher in the PTH group at both 4 and 8 weeks. The modified Wakitani score of the tibial articular surface tissue was significantly higher in the PTH group 8 weeks after surgery.
The results of the macro evaluation are shown in FIG. 5, the results of the meniscal tissue evaluation are shown in FIG. 6, and the results of the tibia tissue evaluation are shown in FIG.
From FIGS. 5 to 7, a tissue morphology closer to that of a normal meniscus, a high meniscal coverage rate, and a chondroprotective effect were observed.
以上より、アキレス腱組織にＰＴＨを注入することで腱組織内に軟骨基質が産生され、ＰＴＨ注入腱を用いた半月再建モデルにおいても軟骨基質が同様に確認された。また、ＰＴＨ注入モデルの方が再建半月板の被覆率が高く、関節軟骨の保護作用が高かった。ＰＴＨ（１－３４）は、軟骨細胞や未分化間葉系細胞に対しては軟骨の後期分化を抑制させる働きが報告されている。腱細胞あるいは腱内の未分化間葉系細胞に対してＰＴＨが作用し、軟骨分化維持させ、半月板様組織が得られる可能性が示唆された。 From the above, a cartilage matrix was produced within the tendon tissue by injecting PTH into the Achilles tendon tissue, and a cartilage matrix was similarly confirmed in the meniscal reconstruction model using the PTH-injected tendon. In addition, the PTH injection model had a higher coverage of the reconstructed meniscus and had a higher protective effect on articular cartilage. PTH (1-34) has been reported to have the effect of suppressing late differentiation of cartilage in chondrocytes and undifferentiated mesenchymal cells. It has been suggested that PTH acts on tendon cells or undifferentiated mesenchymal cells within the tendon, maintains cartilage differentiation, and obtains a meniscal-like tissue.
Claims (12)
提供者から提供された半腱様筋腱組織と、
前記半腱様筋腱組織に注入された請求項１～４のいずれか１項に記載の半月板再生促進剤と、
を含む、半月板再生用材料。 A meniscal regeneration material used to be transplanted into the knee joint in order to regenerate at least a part of the meniscus of the knee joint, the material comprising:
semitendinosus tendon tissue provided by a donor;
The meniscal regeneration promoter according to any one of claims 1 to 4, which is injected into the semitendinosus tendon tissue;
Materials for meniscal regeneration, including:
提供者から提供された半腱様筋腱組織に請求項１～４のいずれか１項に記載の半月板再生促進剤を生体外で注入する工程と、
を含む半月板再生用材料の作製方法。 A method for producing a meniscus regeneration material used to be transplanted into the knee joint in order to regenerate at least a part of the meniscus of the knee joint, the method comprising:
Injecting the meniscal regeneration promoter according to any one of claims 1 to 4 into the semitendinosus tendon tissue provided by a donor in vitro;
A method for producing a material for meniscal regeneration, including:
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