JP2011212437A - Sustained release base material containing dance protein and method for manufacturing sustained release base material - Google Patents
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本発明は、ＤＡＮＣＥタンパク質含有徐放基材及び該徐放基材の製造方法に関する。 The present invention relates to a DANCE protein-containing sustained release substrate and a method for producing the sustained release substrate.
ヒトの生体組織の中でも肺や血管、皮膚等の組織は、弾性、伸縮性に富んだ組織であるが、加齢とともに血管が硬くなる、あるいは皮膚がたるむといった生体組織の弾性が失われていく加齢現象が生じる。これは、生体組織に存在する弾性線維という、ゴムのような柔軟性と弾力を有する線維組織が劣化してしまうためであると考えられている。この弾性線維は、劣化・分解しても再生されることはないとされており、このような弾性線維の再生についての研究が数多くなされている。 Among human biological tissues, tissues such as lungs, blood vessels, and skin are highly elastic and stretchable, but the elasticity of biological tissues such as blood vessels become harder or skin becomes sluggish with age. An aging phenomenon occurs. This is considered to be due to the deterioration of a fibrous tissue having flexibility and elasticity like rubber, which is an elastic fiber present in a living tissue. This elastic fiber is said not to be regenerated even when it is degraded or decomposed, and many studies have been conducted on the regeneration of such elastic fiber.
近年、弾性線維の形成能を有する細胞の無血清培養において、ＤＡＮＣＥ（ｄｅｖｅｌｏｐｍｅｎｔａｌ ａｒｔｅｒｉｅｓ ａｎｄ ｎｅｕｒａｌ ｃｒｅｓｔ ｅｐｉｄｅｒｍａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ（ＥＧＦ）−ｌｉｋｅ；ｆｉｂｕｌｉｎ−５ともいう）という分泌蛋白質（以下、ＤＡＮＣＥタンパク質という）を加えることにより効率よく弾性線維を再生できることが見出された（例えば、特許文献１参照）。 In recent years, a secreted protein (hereinafter referred to as DANCE protein) called DANCE (developmental arteries and neural epidermal growth factor (EGF) -like; also referred to as fibulin-5) is added in serum-free culture of cells having the ability to form elastic fibers. Thus, it has been found that elastic fibers can be efficiently regenerated (see, for example, Patent Document 1).
しかしながら、このＤＡＮＣＥタンパク質を保持できる形態については十分に研究がなされておらず、その取り扱い性が非常に困難であった。 However, the form capable of retaining this DANCE protein has not been sufficiently studied, and its handling is very difficult.
本発明は、ＤＡＮＣＥタンパク質を保持できかつ徐放できる形態である、ＤＡＮＣＥタンパク質含有徐放基材を提供することを目的とする。また、ＤＡＮＣＥタンパク質含有徐放基材の製造方法を提供することを目的とする。 An object of this invention is to provide the DANCE protein containing sustained release base material which is a form which can hold | maintain and can release DANCE protein. Moreover, it aims at providing the manufacturing method of a DANCE protein containing sustained release base material.
本発明者は、上記の課題を解決するために鋭意研究を行った結果、ＤＡＮＣＥタンパク質を保持し、かつ徐放することができる新たな形態を見出した。かかる知見に基づき、さらに研究を重ねて本発明を完成するに至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventor has found a new form capable of retaining and slowly releasing the DANCE protein. Based on this knowledge, further studies have been made and the present invention has been completed.
即ち、本発明は下記のＤＡＮＣＥタンパク質含有徐放基材及びＤＡＮＣＥタンパク質含有徐放基材の製造方法を提供する。
項１．基材及びＤＡＮＣＥタンパク質を含む、ＤＡＮＣＥタンパク質含有徐放基材。
項２．基材が、コラーゲンを含む基材である、項１に記載のＤＡＮＣＥタンパク質含有徐放基材。
項３．基材が、コラーゲンからなる基材、又はコラーゲンとコラーゲン以外の材料とを含む基材である、項１又は２に記載のＤＡＮＣＥタンパク質含有徐放基材。
項４．コラーゲン以外の材料が生体吸収性材料である、項３に記載のＤＡＮＣＥタンパク質含有徐放基材。
項５．コラーゲン以外の材料が乳酸−カプロラクトン共重合体である、項２〜４のいずれかに記載のＤＡＮＣＥタンパク質含有徐放基材。
項６．基材が、コラーゲンスポンジ、コラーゲン粒子、又はコラーゲンフィルムである、項１〜３のいずれかに記載のＤＡＮＣＥタンパク質含有徐放基材。
項７．コラーゲンとＤＡＮＣＥタンパク質溶液を含む溶液を凍結乾燥して得られる、項１、２、３又は６に記載のＤＡＮＣＥタンパク質含有徐放基材。
項８．コラーゲン以外の材料に、コラーゲン溶液にＤＡＮＣＥタンパク質溶液を添加した組成物を含浸させ凍結乾燥して得られる、項２〜５のいずれかに記載のＤＡＮＣＥタンパク質含有徐放基材。
項９．さらにムコ多糖類を含む項１〜８のいずれかに記載のＤＡＮＣＥタンパク質含有徐放基材。
項１０．ムコ多糖類が、コンドロイチン硫酸又はヘパリンである項９に記載のＤＡＮＣＥタンパク質含有徐放基材。
項１１．コラーゲン溶液にＤＡＮＣＥタンパク質溶液を添加した溶液を凍結乾燥する工程を含む、ＤＡＮＣＥタンパク質含有徐放基材の製造方法。
項１２．コラーゲン溶液に、さらにムコ多糖類を添加する項１１に記載の製造方法。
項１３．項１〜１０のいずれかに記載のＤＡＮＣＥタンパク質含有徐放基材によって表面がコーティングされた医療用材料。
項１４．ステント又は人工血管である項１３に記載の医療用材料。
That is, this invention provides the manufacturing method of the following DANCE protein containing sustained release base material and DANCE protein containing sustained release base material.
Item 1. A DANCE protein-containing sustained release substrate comprising a substrate and a DANCE protein.
Item 2. Item 2. The DANCE protein-containing sustained release substrate according to Item 1, wherein the substrate is a substrate containing collagen.
Item 3. Item 3. The DANCE protein-containing sustained release substrate according to Item 1 or 2, wherein the substrate is a substrate made of collagen, or a substrate containing collagen and a material other than collagen.
Item 4. Item 4. The DANCE protein-containing sustained release substrate according to Item 3, wherein the material other than collagen is a bioabsorbable material.
Item 5. Item 5. The DANCE protein-containing sustained release substrate according to any one of Items 2 to 4, wherein the material other than collagen is a lactic acid-caprolactone copolymer.
Item 6. Item 4. The DANCE protein-containing sustained release substrate according to any one of Items 1 to 3, wherein the substrate is a collagen sponge, collagen particles, or a collagen film.
Item 7. Item 7. The DANCE protein-containing sustained release substrate according to Item 1, 2, 3 or 6, obtained by freeze-drying a solution containing collagen and a DANCE protein solution.
Item 8. Item 6. The sustained release substrate containing DANCE protein according to any one of Items 2 to 5, obtained by impregnating a composition other than collagen with a composition obtained by adding a DANCE protein solution to a collagen solution and freeze-drying.
Item 9. Item 9. The DANCE protein-containing sustained release substrate according to any one of Items 1 to 8, further comprising a mucopolysaccharide.
Item 10. Item 10. The DANCE protein-containing sustained release substrate according to Item 9, wherein the mucopolysaccharide is chondroitin sulfate or heparin.
Item 11. A method for producing a DANCE protein-containing sustained release substrate, comprising a step of freeze-drying a solution obtained by adding a DANCE protein solution to a collagen solution.
Item 12. Item 12. The production method according to Item 11, wherein mucopolysaccharide is further added to the collagen solution.
Item 13. Item 11. A medical material whose surface is coated with the DANCE protein-containing sustained release substrate according to any one of Items 1 to 10.
Item 14. Item 14. The medical material according to Item 13, which is a stent or an artificial blood vessel.
本発明のＤＡＮＣＥタンパク質含有徐放基材は、ＤＡＮＣＥタンパク質を保持することができ、さらに添加剤により、ＤＡＮＣＥタンパク質の徐放性スピードのコントロールに優れるものである。 The DANCE protein-containing sustained release substrate of the present invention can retain DANCE protein and is excellent in controlling the sustained release speed of DANCE protein by an additive.
本発明のＤＡＮＣＥタンパク質含有基材は、基材及びＤＡＮＣＥタンパク質を含むものである。 The DANCE protein-containing substrate of the present invention includes a substrate and a DANCE protein.
基材としては、ＤＡＮＣＥタンパク質の保持性及び徐放性に優れることから、コラーゲンを含む基材が好ましく、コラーゲンからなる基材、又はコラーゲンとコラーゲン以外の材料とを含む基材であることがより好ましい。 As a base material, since it is excellent in the retention property and sustained release property of DANCE protein, the base material containing collagen is preferable, and it is more preferably a base material made of collagen or a base material containing collagen and a material other than collagen. preferable.
コラーゲンからなる基材としては、スポンジ（コラーゲンスポンジ）、粒子（コラーゲン粒子）、フィルム（コラーゲンフィルム）、糸（コラーゲン糸）等が挙げられる。 Examples of the base material made of collagen include sponge (collagen sponge), particle (collagen particle), film (collagen film), thread (collagen thread) and the like.
コラーゲン以外の材料としては、生体吸収性材料が挙げられ、具体的には、ポリ乳酸、乳酸−グリコール酸共重合体、乳酸−カプロラクトン共重合体、グリコール酸−カプロラクトン共重合体、ポリジオキサノン、ポリグリコール酸等が挙げられる。これらの中で、乳酸−カプロラクトン共重合体、ポリグリコール酸、グリコール酸−カプロラクトン共重合体が、コラーゲンとの複合化が容易であり、ＤＡＮＣＥタンパク質の保持性及び徐放性に優れる点、加工性に優れる点等から好ましい。 Examples of materials other than collagen include bioabsorbable materials, specifically, polylactic acid, lactic acid-glycolic acid copolymer, lactic acid-caprolactone copolymer, glycolic acid-caprolactone copolymer, polydioxanone, polyglycol. An acid etc. are mentioned. Among these, lactic acid-caprolactone copolymer, polyglycolic acid, glycolic acid-caprolactone copolymer can be easily combined with collagen, has excellent DANCE protein retention and sustained release, and processability It is preferable from the point of being excellent.
コラーゲンスポンジの製造方法としては、コラーゲンとＤＡＮＣＥタンパク質を含む溶液を凍結乾燥して得ることが好ましく、例えば、コラーゲン溶液とＤＡＮＣＥタンパク質溶液を混合した組成物を凍結乾燥して得ることが好ましい。また、コラーゲン粒子の製造方法としては、例えば、前記のコラーゲンスポンジの製造方法によって得られたコラーゲンスポンジを粉砕して得ることができる。さらに、コラーゲンとコラーゲン以外の材料とを含む基材の製造方法としては、コラーゲン以外の材料によって基材を形成させ、その基材に、コラーゲンとＤＡＮＣＥタンパク質を含む組成物を含浸させ凍結乾燥することによって得ることが好ましい。 As a method for producing a collagen sponge, a solution containing collagen and DANCE protein is preferably obtained by lyophilization. For example, a composition obtained by mixing a collagen solution and a DANCE protein solution is preferably obtained by lyophilization. Moreover, as a manufacturing method of a collagen particle, it can obtain by grind | pulverizing the collagen sponge obtained by the manufacturing method of the said collagen sponge, for example. Furthermore, as a method for producing a base material containing collagen and a material other than collagen, the base material is formed of a material other than collagen, and the base material is impregnated with a composition containing collagen and DANCE protein and freeze-dried. It is preferable to obtain by.
基材の形状としては、特に限定されるものではなく、用いられる用途によって適宜設定されるものであるが、例えば、スポンジ状、チューブ状、シート状、粒状、モノフィラメント、糸状、編物、不織布、織物、マルチフィラメント、ステント形状等が挙げられる。 The shape of the substrate is not particularly limited, and is appropriately set depending on the application to be used. For example, sponge, tube, sheet, granular, monofilament, thread, knitted fabric, nonwoven fabric, woven fabric , Multifilament, stent shape and the like.
以下に、本発明のＤＡＮＣＥタンパク質含有徐放基材及びＤＡＮＣＥタンパク質含有徐放基材の製造方法について詳細に説明する。 Below, the manufacturing method of the DANCE protein containing sustained release base material and DANCE protein containing sustained release base material of this invention is demonstrated in detail.
１．コラーゲン溶液
コラーゲンとしては特に限定はなく、牛、豚等の皮膚や腱等に由来するものを用いることができる。抗原性を排除してより安全性を高める観点から、コラーゲンをプロテアーゼやペプシン等の酵素で処理して、テロペプチドをできる限り除去したアテロコラーゲンが好ましい。アテロコラーゲンには、Ｉ〜ＩＶ型があるが、適宜選択することができる。
1. The collagen solution collagen is not particularly limited, and collagen derived from skin such as cows and pigs, tendons and the like can be used. From the viewpoint of enhancing antigenicity by eliminating antigenicity, atelocollagen in which telopeptides are removed as much as possible by treating collagen with an enzyme such as protease or pepsin is preferable. Atelocollagen includes types I to IV, and can be appropriately selected.
コラーゲン溶液の溶媒としては、特に限定されるものではないが、例えば、蒸留水、生理食塩水、リン酸緩衝食塩水（ＰＢＳ）等を挙げることができる。 Although it does not specifically limit as a solvent of a collagen solution, For example, distilled water, physiological saline, phosphate buffered saline (PBS) etc. can be mentioned.
コラーゲン溶液の濃度としては、０．１〜１重量％程度であることが好ましく、０．１〜０．５重量％程度であることがより好ましい。コラーゲン溶液の濃度が前記範囲にあることで、均一なコラーゲン溶液を作製できるため好ましい。 The concentration of the collagen solution is preferably about 0.1 to 1% by weight, and more preferably about 0.1 to 0.5% by weight. It is preferable that the concentration of the collagen solution be in the above range because a uniform collagen solution can be prepared.
２．ＤＡＮＣＥタンパク質溶液
「ＤＡＮＣＥタンパク質」とは、ｆｉｂｕｌｉｎ−５とも呼ばれる分泌蛋白質であり、弾性線維を構成する分子であることが知られている。
2. The DANCE protein solution “DANCE protein” is a secreted protein also called fibulin-5, and is known to be a molecule constituting an elastic fiber.
本発明で用いるＤＡＮＣＥタンパク質は、ヒト由来のヒトＤＡＮＣＥタンパク質、又は、そのオルソログ、あるいはそれらの変異体（ＳＮＰ、ハプロタイプを含む）を含む。 The DANCE protein used in the present invention includes a human-derived human DANCE protein, an ortholog thereof, or a mutant thereof (including SNP and haplotype).
ＤＡＮＣＥタンパク質のオルソログは特に限定されず、例えば任意の動物、好ましくは哺乳動物に由来するものであり得る。哺乳動物としては、例えば、ウシ、ヒツジ、ブタ、ヤギ、サル、ウサギ、ラット、ハムスター、モルモット、マウス等が挙げられる。 The ortholog of the DANCE protein is not particularly limited, and can be derived from any animal, preferably a mammal. Examples of mammals include cattle, sheep, pigs, goats, monkeys, rabbits, rats, hamsters, guinea pigs, mice and the like.
ＤＡＮＣＥタンパク質については、例えば、国際公開第２００６／０８２７６３パンフレット等に記載されている。 About DANCE protein, it describes in the international publication 2006/082763 pamphlet etc., for example.
本発明で用いるＤＡＮＣＥタンパク質溶液のＤＡＮＣＥタンパク質濃度は、特に限定されるものではなく、所望の濃度にすることができるが、例えば、１〜１０,０００ｎｇ／ｍＬ程度であることが好ましく、１０〜８，０００ｎｇ／ｍＬ程度であることがより好ましい。ＤＡＮＣＥタンパク質濃度が前記範囲内にあることで、希釈しすぎることによって生じるＤＡＮＣＥタンパク質の変性や、活性再現性の低下を防ぐことができるため好ましい。 The DANCE protein concentration of the DANCE protein solution used in the present invention is not particularly limited and can be a desired concentration. For example, it is preferably about 1 to 10,000 ng / mL, and 10 to 8 More preferably, it is about 000 ng / mL. It is preferable for the DANCE protein concentration to be within the above-mentioned range since it is possible to prevent the DANCE protein from being denatured and the activity reproducibility from being reduced by excessive dilution.
ＤＡＮＣＥタンパク質溶液に用いる溶媒としては、特に限定されるものではないが、例えば、水（蒸留水）、リン酸緩衝食塩水（ＰＢＳ）、Ｃａｒｍｏｄｙ緩衝液、Ｔｒｉｓ緩衝液等を挙げることができる。これらの中でも、不純物が混入していない点から水（蒸留水）が好ましい。 The solvent used in the DANCE protein solution is not particularly limited, and examples thereof include water (distilled water), phosphate buffered saline (PBS), Carmody buffer, Tris buffer, and the like. Among these, water (distilled water) is preferable because impurities are not mixed.
本発明で用いられるＤＡＮＣＥタンパク質溶液には、ＤＡＮＣＥタンパク質の活性を維持するために、さらに、ゼラチンを含んでいてもよい。 In order to maintain the activity of the DANCE protein, the DANCE protein solution used in the present invention may further contain gelatin.
ゼラチンは、酸性、塩基性両アミノ酸を含む両性電解質であり、等電点４〜６程度のアルカリ処理ゼラチンであることが、ＤＡＮＣＥタンパク質溶液の安定性の点から好ましい。より好ましいゼラチンの等電点は、５〜６程度である。 Gelatin is an ampholyte containing both acidic and basic amino acids, and is preferably alkali-treated gelatin having an isoelectric point of about 4 to 6 from the viewpoint of the stability of the DANCE protein solution. A more preferable isoelectric point of gelatin is about 5-6.
このようなアルカリ処理ゼラチンとしては、市販のものを好適に用いることができ、例えば、ゼラチン粉末（♯２５０、新田ゼラチン（株）製、等電点：５）、ニワトリゼラチン（ニッポンハム（株）製）等を挙げることができる。 As such alkali-treated gelatin, commercially available products can be suitably used. For example, gelatin powder (# 250, manufactured by Nitta Gelatin Co., Ltd., isoelectric point: 5), chicken gelatin (Nippon Ham Co., Ltd.) )) And the like.
３．ＤＡＮＣＥタンパク質含有徐放基材の製造方法
本発明のＤＡＮＣＥタンパク質含有徐放基材は、コラーゲンとＤＡＮＣＥタンパク質を含む溶液を凍結乾燥することにより得ることができる。具体的には、前述のコラーゲン溶液にＤＡＮＣＥタンパク質溶液を添加した組成物を凍結乾燥することにより得ることができる。
3. Production method of DANCE protein-containing sustained release substrate The DANCE protein-containing sustained release substrate of the present invention can be obtained by freeze-drying a solution containing collagen and DANCE protein. Specifically, it can be obtained by lyophilizing a composition obtained by adding a DANCE protein solution to the aforementioned collagen solution.
ＤＡＮＣＥタンパク質溶液の添加量は、コラーゲン溶液全体量に対して、０．１〜５重量％程度であることが好ましく、０．１〜１重量％程度であることがより好ましい。ＤＡＮＣＥタンパク質溶液の添加量が前記範囲内であることにより、コラーゲンが保持できるＤＡＮＣＥタンパク質の量と、徐放されるＤＡＮＣＥタンパク質の量とが好適に釣り合うため好ましい。 The amount of the DANCE protein solution added is preferably about 0.1 to 5% by weight and more preferably about 0.1 to 1% by weight with respect to the total amount of the collagen solution. It is preferable that the amount of the DANCE protein solution added is within the above range because the amount of the DANCE protein that can be retained by the collagen and the amount of the DANCE protein that is slowly released are suitably balanced.
また、本発明においては、コラーゲン溶液にムコ多糖類を添加することが好ましい。ムコ多糖類をコラーゲン溶液に添加して凍結乾燥を行うことで、架橋コラーゲンスポンジを得ることができる。 In the present invention, it is preferable to add mucopolysaccharide to the collagen solution. A cross-linked collagen sponge can be obtained by adding mucopolysaccharide to a collagen solution and freeze-drying it.
ムコ多糖類をコラーゲン溶液に添加した場合、ＤＡＮＣＥタンパク質の徐放速度を制御することができる。これは、ムコ多糖類は親水基を有するため、該ムコ多糖類の添加量により基材の親水基の量をコントロールすることで含水率を制御できるためと考えられている。 When mucopolysaccharide is added to the collagen solution, the sustained release rate of the DANCE protein can be controlled. This is thought to be because the water content can be controlled by controlling the amount of the hydrophilic group of the base material by the amount of the mucopolysaccharide added because the mucopolysaccharide has a hydrophilic group.
また、コラーゲンスポンジを作製する際には、通常、熱処理による架橋が行われるが、本発明において熱架橋を行うとスポンジ中のＤＡＮＣＥタンパク質が失活してしまうため、熱架橋処理を行うことは望ましくない。 Further, when producing a collagen sponge, crosslinking by heat treatment is usually performed. However, if thermal crosslinking is performed in the present invention, DANCE protein in the sponge is deactivated, and therefore it is desirable to perform thermal crosslinking treatment. Absent.
ムコ多糖類としては、例えば、コンドロイチン硫酸、ヘパリン、ヒアルロン酸、デルマタン硫酸、ケラタン硫酸、ヘパラン硫酸等を挙げることができる。これらの中でも、コラーゲンとの溶解性、安価に入手できる点から、コンドロイチン硫酸、ヘパリンが好ましい。 Examples of mucopolysaccharides include chondroitin sulfate, heparin, hyaluronic acid, dermatan sulfate, keratan sulfate, heparan sulfate, and the like. Among these, chondroitin sulfate and heparin are preferable from the viewpoint of solubility with collagen and availability at low cost.
ムコ多糖類の添加方法としては、特に限定されるものではなく、コラーゲン溶液に直接添加することも可能であるが、取扱性の点から、ムコ多糖類を溶媒に溶解した溶液にして、コラーゲン溶液に添加することが好ましい。 The method for adding mucopolysaccharide is not particularly limited, and it can be added directly to the collagen solution. However, from the viewpoint of handling, a solution in which mucopolysaccharide is dissolved in a solvent is used. It is preferable to add to.
ムコ多糖類溶液の濃度としては、０．１〜５重量％程度であることが好ましく、０．１〜１重量％程度であることがより好ましい。ムコ多糖類溶液の濃度が前記範囲にあることで、ムコ多糖類溶液の作製が容易にでき、コラーゲン溶液への溶解性も容易にできるため好ましい。 The concentration of the mucopolysaccharide solution is preferably about 0.1 to 5% by weight, and more preferably about 0.1 to 1% by weight. It is preferable that the concentration of the mucopolysaccharide solution is in the above range because the production of the mucopolysaccharide solution can be facilitated and the solubility in the collagen solution can be facilitated.
ムコ多糖類溶液の溶媒としては、蒸留水、生理食塩水、リン酸緩衝食塩水（ＰＢＳ）等を挙げることができる。 Examples of the solvent for the mucopolysaccharide solution include distilled water, physiological saline, and phosphate buffered saline (PBS).
ムコ多糖類溶液の添加量としては、コラーゲン溶液全体量に対して、１〜５０重量％程度であることが好ましく、１〜３０重量％程度であることがより好ましい。ムコ多糖類溶液の添加量が前記範囲内であることにより、架橋によるスポンジが持つ特有の柔軟性の損失もなく、コラーゲン溶液との溶解性も良いために好ましい。 The addition amount of the mucopolysaccharide solution is preferably about 1 to 50% by weight and more preferably about 1 to 30% by weight with respect to the total amount of the collagen solution. It is preferable that the addition amount of the mucopolysaccharide solution is within the above range, since there is no loss of the flexibility inherent to the sponge due to crosslinking and the solubility in the collagen solution is good.
このようにして調整されたコラーゲン溶液、ＤＡＮＣＥタンパク質溶液及び任意で添加されるムコ多糖類を含む組成物を適当な型枠の中に流延し、−１３５〜−４０℃程度で３０分〜２時間程度凍結後、凍結乾燥することによりＤＡＮＣＥタンパク質含有基材を得る。 A composition containing the collagen solution thus prepared, the DANCE protein solution, and the mucopolysaccharide optionally added is cast into a suitable formwork, and is about −135 to −40 ° C. for 30 minutes to 2 After freezing for about an hour, a DANCE protein-containing substrate is obtained by lyophilization.
得られたＤＡＮＣＥタンパク質含有基材中におけるＤＡＮＣＥタンパク質の含有量は、通常０．００１〜１重量％程度、好ましくは０．０１〜０．８重量％程度である。 The content of DANCE protein in the obtained DANCE protein-containing substrate is usually about 0.001 to 1% by weight, preferably about 0.01 to 0.8% by weight.
本発明のＤＡＮＣＥタンパク質含有徐放基材は、ＤＡＮＣＥタンパク質を保持できる全く新しい形態であり、該基材は、ＤＡＮＣＥタンパク質を徐放することができる。そのため、組織又は器官の再生の足場となるような再生医療用基材、動脈の再生等に用いられる血管再生用基材、陥没やたるみがみられる皮膚の再生用の基材等の用途に用いられる。より具体的には、美容整形用のコラーゲンインプラント、瘢痕治療、人工皮膚、皮膚キット、血管再生基材等の用途に好適に用いられる。 The DANCE protein-containing sustained release substrate of the present invention is a completely new form capable of retaining DANCE protein, and the substrate can release DANCE protein slowly. Therefore, it is used for applications such as a regenerative medical base material that serves as a scaffold for tissue or organ regeneration, a blood vessel regeneration base material that is used for arterial regeneration, a skin regeneration base material that is depressed or sagging, etc. It is done. More specifically, it is suitably used for applications such as cosmetic implants for cosmetic surgery, scar treatment, artificial skin, skin kits, and blood vessel regeneration substrates.
さらに、該基材によって、例えば、ステント、人工血管、人工気管、人工弁等の医療用材料の表面にコーティングさせて用いることも可能である。 Furthermore, the surface of a medical material such as a stent, an artificial blood vessel, an artificial trachea, and an artificial valve can be coated with the base material.
以下、実施例及び比較例を挙げて本発明を説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES Hereinafter, although an Example and a comparative example are given and this invention is demonstrated, this invention is not limited to these Examples.
製造例１（ＤＡＮＣＥ水溶液の調整方法）
凍結乾燥されたＤＡＮＣＥタンパク質（ヒト由来）の粉末４μｇを蒸留水で溶解し、１０μｇ／ｍＬのＤＡＮＣＥタンパク質の水溶液Ａを作製した。
Production Example 1 (Method for preparing DANCE aqueous solution)
4 μg of freeze-dried DANCE protein (human-derived) powder was dissolved in distilled water to prepare an aqueous solution A of 10 μg / mL DANCE protein.
実施例１
コラーゲン（新田ゼラチン（株）製、製品名Ｃｅｌｌｍａｔｒｉｘ ＴｙｐｅＩ−Ｐ）を原料とし、０．３重量％、ｐＨ３のコラーゲン水溶液を調整した。
Example 1
Collagen (manufactured by Nitta Gelatin Co., Ltd., product name Cellmatrix Type IP) was used as a raw material, and a 0.3 wt% collagen aqueous solution having a pH of 3 was prepared.
このコラーゲン水溶液１０ｍＬに、ＤＡＮＣＥタンパク質水溶液Ａ １００μＬを添加した溶液を、凍結乾燥用テフロン(登録商標)シャーレに流し込み、該テフロン(登録商標)シャーレを−１３５℃で凍結し、真空減圧下（０．１ＭＰａ）、−４０℃〜４０℃で１４時間凍結乾燥して、厚さ１ｍｍのＤＡＮＣＥタンパク質含有コラーゲンスポンジを得た。 A solution obtained by adding 100 μL of DANCE protein aqueous solution A to 10 mL of this collagen aqueous solution is poured into a Teflon (registered trademark) petri dish for lyophilization, and the Teflon (registered trademark) petri dish is frozen at −135 ° C. 1 MPa) and lyophilized at −40 ° C. to 40 ° C. for 14 hours to obtain a DANCE protein-containing collagen sponge having a thickness of 1 mm.
実施例２
ヘパリン（和光純薬工業（株）製）を原料とし、０．３重量％のヘパリン水溶液を調整した。
Example 2
Heparin (manufactured by Wako Pure Chemical Industries, Ltd.) was used as a raw material to prepare a 0.3% by weight heparin aqueous solution.
実施例１で調整したコラーゲン水溶液９ｍＬに、ＤＡＮＣＥタンパク質水溶液Ａ １００μＬを添加し、さらに、ヘパリン水溶液１ｍＬを添加した組成物を用いた以外は、実施例１と同様にして、厚さ１ｍｍのＤＡＮＣＥタンパク質含有コラーゲンスポンジを得た。 A DANCE protein having a thickness of 1 mm was prepared in the same manner as in Example 1 except that 100 μL of DANCE protein aqueous solution A was added to 9 mL of the collagen aqueous solution prepared in Example 1, and further 1 mL of heparin aqueous solution was added. A collagen sponge was obtained.
実施例３
コンドロイチン硫酸（和光純薬工業（株）製）を原料とし、０．３重量％のコンドロイチン硫酸水溶液を調整した。
Example 3
Using chondroitin sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) as a raw material, a 0.3 wt% chondroitin sulfate aqueous solution was prepared.
実施例１で調整したコラーゲン水溶液９ｍＬに、ＤＡＮＣＥタンパク質水溶液Ａ １００μＬを添加し、さらに、コンドロイチン溶液１ｍＬを添加した組成物を用いた以外は、実施例１と同様にして、厚さ１ｍｍのＤＡＮＣＥタンパク質含有コラーゲンスポンジを得た。 A DANCE protein having a thickness of 1 mm was prepared in the same manner as in Example 1 except that 100 μL of DANCE protein aqueous solution A was added to 9 mL of the collagen aqueous solution prepared in Example 1, and further 1 mL of chondroitin solution was used. A collagen sponge was obtained.
比較例１
実施例１で得られたスポンジを、真空下１１０℃で２４時間加熱処理することにより熱架橋を行った。
Comparative Example 1
The sponge obtained in Example 1 was heat-crosslinked by heat treatment at 110 ° C. for 24 hours under vacuum.
比較例２
ゼラチン粉末（新田ゼラチン（株）製、製品名 ＃250）を蒸留水に溶解し、０．３重量％、ゼラチン水溶液を調整した。このゼラチン水溶液１０ｍＬに、ＤＡＮＣＥタンパク質水溶液Ａ １００μＬを添加した溶液を、凍結乾燥用テフロン(登録商標)シャーレに流し込み、該テフロン(登録商標)シャーレを−１３５℃で凍結し、真空減圧下（０．１ＭＰａ）、−４０℃〜４０℃で１４時間凍結乾燥して、厚さ１ｍｍのＤＡＮＣＥタンパク質含有ゼラチンスポンジを得た。
（試験）
実施例１〜３及び比較例１、２で得られた基材を、リン酸緩衝食塩水（ＰＢＳ）１０ｍＬに浸漬し、３７℃で保管した。浸漬後１時間、１日、３日、７日、１４日、２１日、２８日に、ＰＢＳ中に放出されたＤＡＮＣＥタンパク質の濃度を、ＥＬＩＳＡ法により測定した。抗体は、マウス抗ＤＡＮＣＥタンパク質ＩｇＧを用いた。測定キット（製造元：エヌビィ健康研究所）を用い、実施例１〜３、比較例１〜２のＤＡＮＣＥ溶液のＤＡＮＣＥ濃度を測定した。その結果を下記表１に示す。
Comparative Example 2
Gelatin powder (manufactured by Nitta Gelatin Co., Ltd., product name # 250) was dissolved in distilled water to prepare a 0.3 wt% gelatin aqueous solution. A solution obtained by adding 100 μL of DANCE protein aqueous solution A to 10 mL of this gelatin aqueous solution was poured into a Teflon (registered trademark) petri dish for freeze-drying, and the Teflon (registered trademark) petri dish was frozen at −135 ° C. 1 MPa), and freeze-dried at −40 ° C. to 40 ° C. for 14 hours to obtain a DANCE protein-containing gelatin sponge having a thickness of 1 mm.
(test)
The base materials obtained in Examples 1 to 3 and Comparative Examples 1 and 2 were immersed in 10 mL of phosphate buffered saline (PBS) and stored at 37 ° C. The concentration of DANCE protein released in PBS was measured by ELISA method 1 hour, 1 day, 3 days, 7 days, 14 days, 21 days and 28 days after immersion. Mouse anti-DANCE protein IgG was used as the antibody. Using a measurement kit (manufacturer: NU Health Laboratory), the DANCE concentrations of the DANCE solutions of Examples 1 to 3 and Comparative Examples 1 and 2 were measured. The results are shown in Table 1 below.
実施例１〜３から明らかなように、これら３種の基材は、ＤＡＮＣＥタンパク質の徐放性に優れ、ムコ多糖溶液を添加した実施例２、３では、添加しなかった場合（実施例１）よりも早くＤＡＮＣＥタンパク質が放出された。これは、ムコ多糖が親水基を有することにより、スポンジ基材の含水率が高くなったためと考えられる。 As is clear from Examples 1 to 3, these three types of base materials are excellent in sustained release of DANCE protein, and in Examples 2 and 3 to which a mucopolysaccharide solution was added (Example 1). DANCE protein was released earlier than This is presumably because the moisture content of the sponge base material increased due to the mucopolysaccharide having a hydrophilic group.
また、熱架橋を行った比較例１では、ＤＡＮＣＥタンパク質が測定されなかった。これは、熱架橋により、ＤＡＮＣＥタンパク質が失活したためと考えられる。 In Comparative Example 1 in which thermal crosslinking was performed, DANCE protein was not measured. This is presumably because the DANCE protein was inactivated by thermal crosslinking.
ゼラチンを用いた比較例２では、浸漬後１日以内にほとんどのＤＡＮＣＥタンパク質が溶出してしまい、徐放することができなかった。これは、ゼラチンスポンジが直ちに溶解してしまったためであると考えられる。 In Comparative Example 2 using gelatin, most of the DANCE protein was eluted within one day after immersion, and could not be released slowly. This is probably because the gelatin sponge immediately dissolved.
コラーゲン溶液とＤＡＮＣＥタンパク質を混合した水溶液を凍結乾燥することによって、ＤＡＮＣＥタンパク質を徐放することが可能な基材を作製することが可能になった。 By freeze-drying an aqueous solution in which a collagen solution and a DANCE protein are mixed, it is possible to produce a substrate capable of sustained release of the DANCE protein.
実施例４
乳酸とカプロラクトン共重合体からなる人工血管基材を直径９ｍｍに打ち抜き、０．３％コラーゲン溶液１ｍｌ（ｐＨ３)にＤＡＮＣＥ溶液１００μｌ（１００μｇ／ｍｌ）を添加した溶液を５００μｌ滴下し、−４０℃〜４０℃で１２時間凍結乾燥を行い、人工血管基材にＤＡＮＣＥタンパク質含有徐放剤を一体化させた。
Example 4
An artificial blood vessel substrate made of lactic acid and caprolactone copolymer was punched out to a diameter of 9 mm, and 500 μl of a solution obtained by adding 100 μl of DANCE solution (100 μg / ml) to 1 ml of 0.3% collagen solution (pH 3) was dropped, and the temperature was −40 ° C. Lyophilization was carried out at 40 ° C. for 12 hours, and the DANCE protein-containing sustained release agent was integrated with the artificial blood vessel substrate.
その後、リン酸緩衝食塩水（ＰＢＳ）１０ｍＬに浸漬し、３７℃で保管した。浸漬後１時間、１日、３日、７日に、ＰＢＳ中に放出されたＤＡＮＣＥタンパク質の濃度を、ＥＬＩＳＡ法により測定した。抗体は、マウス抗ＤＡＮＣＥタンパク質ＩｇＧを用いた。測定キット（製造元：エヌビィ健康研究所）を用い、ＤＡＮＣＥ溶液のＤＡＮＣＥ濃度を測定した。測定結果を表２に示す。 Then, it was immersed in 10 mL of phosphate buffered saline (PBS) and stored at 37 ° C. The concentration of DANCE protein released in PBS was measured by ELISA method for 1 hour, 1 day, 3 days, and 7 days after immersion. Mouse anti-DANCE protein IgG was used as the antibody. The DANCE concentration of the DANCE solution was measured using a measurement kit (manufacturer: NU Health Laboratory). The measurement results are shown in Table 2.
実施例５
コラーゲン（新田ゼラチン（株）製、製品名Ｃｅｌｌｍａｔｒｉｘ ＴｙｐｅＩ−Ｐ）を原料とし、０．３重量％、ｐＨ３のコラーゲン水溶液を調整した。
Example 5
Collagen (manufactured by Nitta Gelatin Co., Ltd., product name Cellmatrix Type IP) was used as a raw material, and a 0.3 wt% collagen aqueous solution having a pH of 3 was prepared.
このコラーゲン水溶液１０ｍＬに、ＤＡＮＣＥタンパク質水溶液Ａ １００μＬを添加した溶液を、凍結乾燥用テフロン（登録商標）シャーレに流し込み、該テフロン（登録商標）シャーレを−１３５℃で凍結し、真空減圧下（０．１ＭＰａ）、−４０℃〜４０℃で１４時間凍結乾燥して、厚さ１ｍｍのＤＡＮＣＥタンパク質含有コラーゲンスポンジを得た。 A solution obtained by adding 100 μL of DANCE protein aqueous solution A to 10 mL of this collagen aqueous solution is poured into a Teflon (registered trademark) petri dish for freeze-drying, and the Teflon (registered trademark) petri dish is frozen at −135 ° C. 1 MPa) and lyophilized at −40 ° C. to 40 ° C. for 14 hours to obtain a DANCE protein-containing collagen sponge having a thickness of 1 mm.
このスポンジを凍結させ、小型粉砕機を用いて粉砕し、ＤＡＮＣＥタンパク質含有コラーゲン粒子を得た。 The sponge was frozen and pulverized using a small pulverizer to obtain DANCE protein-containing collagen particles.
その後、リン酸緩衝食塩水（ＰＢＳ）１０ｍＬに浸漬し、３７℃で保管した。浸漬後１時間、１日、３日、７日に、ＰＢＳ中に放出されたＤＡＮＣＥタンパク質の濃度を、ＥＬＩＳＡ法により測定した。抗体は、マウス抗ＤＡＮＣＥタンパク質ＩｇＧを用いた。測定キット（製造元：エヌビィ健康研究所）を用い、ＤＡＮＣＥ溶液のＤＡＮＣＥ濃度を測定した。測定結果を表２に示す。 Then, it was immersed in 10 mL of phosphate buffered saline (PBS) and stored at 37 ° C. The concentration of DANCE protein released in PBS was measured by ELISA method for 1 hour, 1 day, 3 days, and 7 days after immersion. Mouse anti-DANCE protein IgG was used as the antibody. The DANCE concentration of the DANCE solution was measured using a measurement kit (manufacturer: NU Health Laboratory). The measurement results are shown in Table 2.
実施例６
乳酸とカプロラクトン共重合体からなる縫合糸（直径０．５ｍｍ）を編み、内径５ｍｍの吸収性のステントを作製した。
Example 6
An absorptive stent having an inner diameter of 5 mm was produced by knitting a suture (diameter 0.5 mm) made of lactic acid and a caprolactone copolymer.
厚さ０．５ｍｍのポリグリコール酸不織布を作製し、コラーゲン水溶液１０ｍＬに、ＤＡＮＣＥタンパク質水溶液Ａ １００μＬを添加した溶液を、ガラス板にキャストし、一定の厚さになるように広げ、その上に上記で作製した不織布を置き、一晩風乾させ、ＤＡＮＣＥタンパク質含有コラーゲンと不織布が一体化したものを作製した。 A polyglycolic acid non-woven fabric having a thickness of 0.5 mm was prepared, and a solution obtained by adding 100 μL of DANCE protein aqueous solution A to 10 mL of collagen aqueous solution was cast on a glass plate and spread to a certain thickness. The non-woven fabric prepared in (1) was placed and air-dried overnight to produce an integrated DANCE protein-containing collagen and non-woven fabric.
吸収性ステントの内周とほぼ同じ大きさにカットしたポリグリコール酸不織布をステント内部に入れ、ぴったりくっつくように円筒状とし、周りからジオキサンを塗布し、風乾させた。その結果、吸収性ステントとポリグリコール酸不織布とＤＡＮＣＥタンパク質含有コラーゲンフィルムが一体となったステントが得られた。 A polyglycolic acid non-woven fabric cut to approximately the same size as the inner circumference of the absorbent stent was placed inside the stent, formed into a cylindrical shape so as to adhere closely, and dioxane was applied from the periphery and allowed to air dry. As a result, a stent in which an absorbent stent, a polyglycolic acid nonwoven fabric, and a DANCE protein-containing collagen film were integrated was obtained.
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|---|---|---|---|---|
| JP2011193931A (en) * | 2010-03-17 | 2011-10-06 | Gunze Ltd | Base material for dance protein-containing tissue regeneration |
| JP2017537934A (en) * | 2014-12-07 | 2017-12-21 | ヴェッスル セラピューティクス リミテッド | Use of fibulin-5 to treat keloid scars |
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| CN108837181A (en) * | 2018-06-20 | 2018-11-20 | 罗国球 | A kind of bionical collagem membrane and preparation method thereof of high intensity |
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| JP2011193931A (en) * | 2010-03-17 | 2011-10-06 | Gunze Ltd | Base material for dance protein-containing tissue regeneration |
| JP2017537934A (en) * | 2014-12-07 | 2017-12-21 | ヴェッスル セラピューティクス リミテッド | Use of fibulin-5 to treat keloid scars |
| WO2018056018A1 (en) * | 2016-09-21 | 2018-03-29 | グンゼ株式会社 | Method for producing porous substrate comprising bioabsorbable polymer that contains heparin, porous substrate comprising bioabsorbable polymer that contains heparin, and artificial blood vessel |
| CN109715225A (en) * | 2016-09-21 | 2019-05-03 | 郡是株式会社 | Manufacturing method containing heparin and comprising the high molecular porous substrate of bioresorbable, containing heparin and include the high molecular porous substrate of bioresorbable and artificial blood vessel |
| CN109715225B (en) * | 2016-09-21 | 2021-06-15 | 郡是株式会社 | Porous base material containing heparin and containing bioabsorbable polymer, method for producing same, and artificial blood vessel |
| US11273237B2 (en) | 2016-09-21 | 2022-03-15 | Gunze Limited | Method for producing porous substrate comprising bioabsorbable polymer that contains heparin, porous substrate comprising bioabsorbable polymer that contains heparin, and artificial blood vessel |
| CN108837181A (en) * | 2018-06-20 | 2018-11-20 | 罗国球 | A kind of bionical collagem membrane and preparation method thereof of high intensity |
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