JPH083199A - Collagen-chondrocyte complex - Google Patents
Collagen-chondrocyte complexInfo
- Publication number
- JPH083199A JPH083199A JP6156754A JP15675494A JPH083199A JP H083199 A JPH083199 A JP H083199A JP 6156754 A JP6156754 A JP 6156754A JP 15675494 A JP15675494 A JP 15675494A JP H083199 A JPH083199 A JP H083199A
- Authority
- JP
- Japan
- Prior art keywords
- collagen
- cartilage
- bfgf
- cell
- tissue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Materials For Medical Uses (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、塩基性線維芽細胞増殖
因子（Basic Fibroblast Growth Factor、以下ｂＦＧＦ
と略称する）を含有するコラーゲン−軟骨細胞複合体お
よび該複合体からなる軟骨組織再生治療材に関するもの
である。BACKGROUND OF THE INVENTION The present invention relates to basic fibroblast growth factor (hereinafter referred to as bFGF).
(Hereinafter abbreviated as ") -containing collagen-chondrocyte complex, and a cartilage tissue regeneration treatment material comprising the complex.
【０００２】[0002]
【従来の技術】ｉｎ ｖｉｔｒｏで軟骨細胞を増殖させ
て軟骨組織を再生できれば、再建外科においてきわめて
有用である。しかし、軟骨細胞は通常の培地を用いたｉ
ｎ ｖｉｔｒｏ培養下では盛んに増殖するにもかかわら
ず、軟骨組織には分化しない。そのため、軟骨組織の再
建という作業は生体に託したほうが近道のように思われ
る。すでに、生体内分解吸収性合成高分子であるポリ乳
酸を足場としてそれに軟骨細胞を付着させてヌードマウ
ス体内に埋め込むことによって軟骨組織の再建ができた
という報告はあるが、これらは数カ月以上の長時間を要
している。2. Description of the Related Art It is extremely useful in reconstructive surgery if cartilage cells can be regenerated by proliferating chondrocytes in vitro. However, chondrocytes were i
Although it proliferates vigorously in n vitro culture, it does not differentiate into cartilage tissue. Therefore, it seems that the task of rebuilding cartilage tissue should be entrusted to the living body. It has already been reported that cartilage tissue could be reconstructed by using polylactic acid, which is a biodegradable and absorbable synthetic polymer, as a scaffold to attach chondrocytes to the scaffold and implant it in the body of nude mice. It takes time.
【０００３】[0003]
【発明が解決しようとする課題】本発明者らは、ｂＦＧ
Ｆを含有するコラーゲン−軟骨細胞複合体が、わずか２
週間という短期間で効率よく、生体内での軟骨組織再生
に著明な効果を示すことを見出し、本発明を完成した。DISCLOSURE OF THE INVENTION The present inventors have found that bFG
Only 2 F-containing collagen-chondrocyte complexes
The present invention has been completed based on the finding that it has a remarkable effect on cartilage tissue regeneration in vivo efficiently in a short period of weeks.
【０００４】[0004]
【課題を解決するための手段】本発明のｂＦＧＦ含有コ
ラーゲン−軟骨細胞複合体は、生体適合性がよく、生体
内で軟骨細胞を増殖させ、かつ軟骨組織の再生を促進
し、多くの軟骨疾患に適用できる。The bFGF-containing collagen-chondrocyte complex of the present invention has good biocompatibility, proliferates chondrocytes in vivo, promotes regeneration of cartilage tissue, and causes many cartilage diseases. Applicable to
【０００５】本発明のｂＦＧＦ含有コラーゲン−軟骨細
胞複合体におけるｂＦＧＦは、脳下垂体、脳、網膜、黄
体、副腎、腎、胎盤、前立腺、胸腺などの臓器より抽出
されるもの、組換えＤＮＡ技術などの遺伝子工学的手法
で製造されるもの、さらにこれらの修飾体であって線維
芽細胞増殖因子として作用し得るものを含む。The bFGF in the collagen-chondrocyte complex containing bFGF of the present invention is extracted from organs such as pituitary gland, brain, retina, corpus luteum, adrenal gland, kidney, placenta, prostate and thymus, recombinant DNA technology. And those modified by these, which are capable of acting as fibroblast growth factors.
【０００６】ｂＦＧＦの修飾体としては、例えば上記の
抽出により得られたまたは遺伝子工学的手法で得られた
ｂＦＧＦのアミノ酸配列においてアミノ酸が付加された
もの、アミノ酸の一部が他のアミノ酸で置換されたも
の、またはアミノ酸の一部が欠損したものなどが挙げら
れる。本発明においては、これらのｂＦＧＦまたはその
修飾体は単独で用いてもよいし、これらの混合物として
用いてもよい。The modified bFGF is, for example, an amino acid added to the amino acid sequence of bFGF obtained by the above extraction or obtained by a genetic engineering method, or a part of the amino acid is replaced with another amino acid. And those lacking a part of amino acids. In the present invention, these bFGF or modified forms thereof may be used alone or as a mixture thereof.
【０００７】上記ｂＦＧＦとしては、好ましくは、例え
ばＷＯ８７／０１７２８（特表昭６３−５００８４３号
公報）、ＷＯ８９／０４８３２（特表平２−５０４４６
８号公報）、ＷＯ８６／０７５９５（特表昭６３−５０
００３６号公報）、ＷＯ８７／０３８８５（特表昭６３
−５０１９５３号公報）、欧州特許出願公開第２３７９
６６号（特開昭６３−２２６２８７号公報）、欧州特許
出願公開第２８１８２２号（特開平２−１９３号公
報）、欧州特許出願公開第３２６９０７号（特開平２−
２０９８９４号公報）、欧州特許出願公開第３９４９５
１号（特開平３−６１４９４号公報）、欧州特許出願公
開第４９３７３７号（特開平５−１２４９７５号公報）
などに記載のものが挙げられる。The above-mentioned bFGF is preferably, for example, WO87 / 01728 (Japanese Patent Publication No. 63-500843), WO89 / 04832 (Japanese Patent Publication No. 2-50446).
No. 8), WO86 / 07595 (Special Table Sho 63-50)
0036), WO87 / 03885 (Sho 63
-501953), European Patent Application Publication No. 2379.
No. 66 (Japanese Patent Laid-Open No. 226287/1988), European Patent Application Publication No. 281822 (Japanese Patent Laid-Open No. 2-193), and European Patent Application Publication No. 326907 (Japanese Patent Laid-Open No. 2-207907).
209894), European Patent Application Publication No. 39495.
No. 1 (JP-A-3-61494), EP-A-493737 (JP-A-5-124975).
And the like.
【０００８】これらのｂＦＧＦのうち、ＷＯ８７／０１
７２８に記載の遺伝子工学的手法で製造した配列表にお
ける配列番号１の１５４個のアミノ酸配列を有するポリ
ペプチドおよび配列番号２の１５３個のアミノ酸配列を
有するポリペプチドが、安定性および材料として必要な
量を常時供給することが容易であるという点から特に好
ましい。Among these bFGF, WO87 / 01
The polypeptide having the 154 amino acid sequence of SEQ ID NO: 1 and the polypeptide having the 153 amino acid sequence of SEQ ID NO: 2 in the sequence listing produced by the genetic engineering method described in 728 are required as stability and materials. It is particularly preferable because it is easy to constantly supply the amount.
【０００９】一方、コラーゲンは皮膚などのＩ型コラー
ゲンからなる動物組織より調製したのち、種々の形状、
例えばスポンジ状、ゲル状、綿状、さらには実際に適用
する耳や鼻などの形状に加工し使用できる。特に、スポ
ンジ状が好ましく、そのスポンジの孔は径１００〜５０
０μｍ、密度は０．０１ｇ／ｃｍ3 が好ましい。また、
必要に応じ、グルタルアルデヒドや熱を加えることによ
り架橋を導入できる。On the other hand, collagen was prepared from animal tissue composed of type I collagen such as skin, and then prepared in various shapes.
For example, it can be used after being processed into a sponge shape, a gel shape, a cotton shape, or an ear or nose shape to be actually applied. In particular, a sponge shape is preferable, and the pores of the sponge have a diameter of 100 to 50.
The density is preferably 0 μm and the density is preferably 0.01 g / cm 3 . Also,
If necessary, crosslinking can be introduced by adding glutaraldehyde or heat.
【００１０】スポンジ状のｂＦＧＦ含有コラーゲン−軟
骨細胞複合体を調製するには、該コラーゲンスポンジを
使用目的に応じた大きさに調製した後、あるいは適当な
大きさに調製した後、例えば、ｂＦＧＦの生理食塩水溶
液またはリン酸緩衝生理食塩水溶液１〜１０００μｇ／
ｍｌを１２〜２４時間、０℃〜室温にて含浸し、さらに
軟骨細胞分散液を調製したｂＦＧＦ含有コラーゲンスポ
ンジに注入して１〜２時間静置することにより、ｂＦＧ
Ｆを含有したコラーゲン−軟骨細胞複合体を得ることが
できる。その他の形状の複合体も同様の方法にて調製で
きる。本複合体を調製する際のｂＦＧＦの濃度は、使用
目的、使用部位、疾病の程度にて、適宜変えられるが、
好ましくは１〜１０００μｇ／ｍｌである。本複合体を
例えば耳の軟骨の再建に用いる場合は、ｂＦＧＦ濃度１
μｇ／ｍｌに含浸し軟骨細胞分散液を注入した本発明複
合体を使用部位へ埋め込ことにより再建できる。To prepare a sponge-like collagen-chondrocyte complex containing bFGF, the collagen sponge is prepared in a size suitable for the purpose of use, or after being prepared in an appropriate size, for example, bFGF Physiological saline solution or phosphate buffered saline solution 1-1000 μg /
ml for 12 to 24 hours at 0 ° C. to room temperature, and the chondrocyte dispersion liquid was further injected into the prepared bFGF-containing collagen sponge and left standing for 1 to 2 hours.
A collagen-chondrocyte complex containing F can be obtained. Complexes of other shapes can be prepared in the same manner. The concentration of bFGF at the time of preparing this complex can be appropriately changed depending on the purpose of use, site of use, degree of disease,
It is preferably 1 to 1000 μg / ml. When this complex is used to reconstruct cartilage of the ear, for example, bFGF concentration 1
The complex of the present invention impregnated with μg / ml and injected with the chondrocyte dispersion liquid can be reconstructed by embedding it in the site of use.
【００１１】[0011]
【実施例】細胞培養の足場として、Ｉ型コラーゲンスポ
ンジを用いた。スポンジを円形（直径９ｍｍ）に打ち抜
いて二枚重ね合わせ、周囲を縫い合わせて袋状とした。
７０％エタノールに１日間浸漬した後、１０００μｇ／
ｍｌのｂＦＧＦ溶液に１日間浸漬し、コラーゲンスポン
ジ内に軟骨細胞分散液を注射器で１×１０6 個注入し、
２時間静置して細胞を付着させ、ｂＦＧＦ含有コラーゲ
ン−軟骨細胞複合体を得た。なお、軟骨細胞は細切した
ラット肋軟骨をコラゲナーゼ処理し、培養シャーレ上で
約１ヶ月間静置培養して初代軟骨細胞株を得た。Example A type I collagen sponge was used as a scaffold for cell culture. A sponge was punched out into a circular shape (diameter 9 mm), two pieces were overlapped, and the periphery was sewn to form a bag.
After soaking in 70% ethanol for 1 day, 1000 μg /
Immerse in 1 ml of bFGF solution for 1 day, inject 1 × 10 6 chondrocyte dispersion liquid into collagen sponge with a syringe,
The cells were allowed to adhere by standing for 2 hours to obtain a bFGF-containing collagen-chondrocyte complex. The chondrocytes were obtained by subjecting chopped rat costal cartilage to collagenase treatment and culturing statically on a culture dish for about 1 month to obtain a primary chondrocyte cell line.
【００１２】[0012]
【試験例】実施例で調製したコラーゲン−軟骨細胞複合
体さらに対照として未処理のコラーゲンスポンジ（以下
対照１と略記する）およびｂＦＧＦを含有しないコラー
ゲン−軟骨細胞複合体（以下対照２と略記する）をそれ
ぞれヌードマウスの背部皮下に埋め込み、所定期間埋入
後にサンプルを取り出し、常法に従って切片を作成後、
ヘマトキシリン−エオジン、サフラニンＯ−ファストグ
リーン、およびチオニン染色を行い、光学顕微鏡にて観
察した。[Test Example] Collagen-chondrocyte complex prepared in Example Further, as a control, an untreated collagen sponge (hereinafter abbreviated as Control 1) and a collagen-chondrocyte complex not containing bFGF (hereinafter abbreviated as Control 2). Embedded in the dorsal skin of each nude mouse, and after taking out the sample for a predetermined period of time, take out the sample and prepare a section according to a conventional method,
Hematoxylin-eosin, safranine O-fast green, and thionine staining were performed and observed with an optical microscope.
【００１３】各試料を埋め込んだ後、ヌードマウスの外
観を４週間観察した。対照１の試料を埋め込んでから４
週間後には外見上ほとんど吸収されていた。対照２の試
料を埋め込んでから２週間後までは軟骨組織は認められ
なかったが、４週間後の組織切片を観察すると軟骨特有
の組織の形成されているのが認められたが、それは埋め
込んだ試料のごく一部のみであった。本発明の複合体を
埋め込んでから１週間後では、軟骨組織は認められなか
ったが、チオニンの異染色から、細胞間に軟骨プロテオ
グリカンの産生されているのが認められた。試料内には
多数の毛細血管も新生していた。４週間後では、埋め込
んだ試料のほぼ全面に軟骨組織が観察される。また、こ
の場合、試料全体を白色の塊としてマウスから取り出す
ことができた。After embedding each sample, the appearance of nude mice was observed for 4 weeks. 4 after embedding control 1 sample
After a week, it was almost completely absorbed. Cartilage tissue was not observed until 2 weeks after the control 2 sample was implanted, but when a tissue section was observed 4 weeks later, formation of tissue specific to cartilage was observed, which was embedded. Only a small portion of the sample. One week after implanting the complex of the present invention, no cartilage tissue was observed, but it was confirmed that cartilage proteoglycan was produced between the cells from the heterostaining of thionine. A large number of capillaries were also formed in the sample. After 4 weeks, cartilage tissue is observed on almost the entire surface of the embedded sample. Also, in this case, the entire sample could be removed from the mouse as a white mass.
【００１４】以上の結果から、コラーゲンスポンジに軟
骨細胞を付着させてヌードマウスに埋め込むことによっ
て軟骨組織の再生されることがわかった。さらに、ｂＦ
ＧＦを加えることによって組織の再生量と速度を格段に
高めることが認められた。From the above results, it was found that cartilage tissue was regenerated by attaching chondrocytes to a collagen sponge and embedding it in nude mice. Furthermore, bF
It was found that the addition of GF markedly increased the amount and rate of tissue regeneration.
【００１５】[0015]
【発明の効果】本発明のｂＦＧＦ含有コラーゲン−軟骨
細胞複合体は、生体適合性がよく、生体内で軟骨細胞を
増殖させ、かつ軟骨組織の再生を促進し、多くの軟骨疾
患に適用できる。INDUSTRIAL APPLICABILITY The bFGF-containing collagen-chondrocyte complex of the present invention has good biocompatibility, proliferates chondrocytes in vivo, promotes regeneration of cartilage tissue, and can be applied to many cartilage diseases.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ０７Ｋ 14/50 8318−4Ｈ 19/00 8318−4Ｈ // Ａ６１Ｋ 38/17 ＡＢＪ 38/22 ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI Technical display area C07K 14/50 8318-4H 19/00 8318-4H // A61K 38/17 ABJ 38/22
Claims (4)
−軟骨細胞複合体。1. A collagen-chondrocyte complex containing basic fibroblast growth factor.
とする請求項１記載のコラーゲン−軟骨細胞複合体2. The collagen-chondrocyte complex according to claim 1, wherein the collagen is sponge-like.
とする請求項１記載のコラーゲン−軟骨細胞複合体。3. The collagen-chondrocyte complex according to claim 1, wherein the chondrocytes are human chondrocytes.
細胞複合体からなる軟骨組織再生治療材。4. A cartilage tissue regeneration treatment material comprising the collagen-chondrocyte complex according to any one of claims 1 to 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6156754A JPH083199A (en) | 1994-06-15 | 1994-06-15 | Collagen-chondrocyte complex |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6156754A JPH083199A (en) | 1994-06-15 | 1994-06-15 | Collagen-chondrocyte complex |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH083199A true JPH083199A (en) | 1996-01-09 |
Family
ID=15634592
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6156754A Pending JPH083199A (en) | 1994-06-15 | 1994-06-15 | Collagen-chondrocyte complex |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH083199A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4952521A (en) * | 1985-04-26 | 1990-08-28 | Fujitsu Limited | Process for fabricating a semiconductor device with selective growth of a metal silicide |
| WO1997043312A1 (en) * | 1996-05-09 | 1997-11-20 | Deutsche Gelatine-Fabriken Stoess Ag | Gelatine consisting substantially of polypeptides of the collagens ii, vi, ix, x, xi, xii and xiv, process for preparing the same and use thereof |
| JP2001519210A (en) * | 1997-10-10 | 2001-10-23 | エド・ガイストリヒ・ゼーネ・アクチエンゲゼルシャフト・フューア・ヒェーミシェ・インドゥストリー | Membrane used for tissue regeneration induction |
| JP2004255110A (en) * | 2003-02-27 | 2004-09-16 | Sangaku Renkei Kiko Kyushu:Kk | Artificial lymph node |
-
1994
- 1994-06-15 JP JP6156754A patent/JPH083199A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4952521A (en) * | 1985-04-26 | 1990-08-28 | Fujitsu Limited | Process for fabricating a semiconductor device with selective growth of a metal silicide |
| WO1997043312A1 (en) * | 1996-05-09 | 1997-11-20 | Deutsche Gelatine-Fabriken Stoess Ag | Gelatine consisting substantially of polypeptides of the collagens ii, vi, ix, x, xi, xii and xiv, process for preparing the same and use thereof |
| JP2001519210A (en) * | 1997-10-10 | 2001-10-23 | エド・ガイストリヒ・ゼーネ・アクチエンゲゼルシャフト・フューア・ヒェーミシェ・インドゥストリー | Membrane used for tissue regeneration induction |
| JP4819995B2 (en) * | 1997-10-10 | 2011-11-24 | エド・ガイストリヒ・ゼーネ・アクチエンゲゼルシャフト・フューア・ヒェーミシェ・インドゥストリー | Membrane used to induce tissue regeneration |
| JP2004255110A (en) * | 2003-02-27 | 2004-09-16 | Sangaku Renkei Kiko Kyushu:Kk | Artificial lymph node |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2101556C (en) | Growth factor containing matrix for the treatment of cartilage lesions | |
| KNAPP et al. | Injectable collagen for soft tissue augmentation | |
| Halberstadt et al. | A hydrogel material for plastic and reconstructive applications injected into the subcutaneous space of a sheep | |
| EP1076533B1 (en) | Guided development and support of hydrogel-cell compositions | |
| De Vries et al. | Dermal regeneration in native non‐cross‐linked collagen sponges with different extracellular matrix molecules | |
| KR101422689B1 (en) | Cell therapy product for cartilage damage comprising collagen, hyaluronic acid derivative and mammalian umbilical cord-derived stem cells | |
| JP3559566B2 (en) | Use of autologous dermal fibroblasts for repair of skin and soft tissue defects | |
| Bauer et al. | Long-term result of vocal cord augmentation with autogenous fat | |
| US20040176855A1 (en) | Decellularized liver for repair of tissue and treatment of organ deficiency | |
| WO1999052356A1 (en) | Creation of three-dimensional tissues | |
| JPH04505717A (en) | In vivo cartilage generation from cell cultures | |
| WO1985004185A1 (en) | Process of and material for stimulating growth of cartilage and bony tissue at anatomical sites | |
| JPH01256967A (en) | Injectable soft tissue prosthesis derived from placenta and its manufacturing method | |
| WO2003084980A2 (en) | Peptide amphiphile solutions and self assembled peptide nanofiber networks | |
| JPH04500954A (en) | Collagen matrix and its production method for wound healing | |
| HK1006416B (en) | Growth factor containing matrix for the treatment of cartilage lesions | |
| JP2510503B2 (en) | Brain-derived cell growth factor | |
| CN1148230C (en) | Preparation of compound artificial skin | |
| JP2001509064A (en) | Augmentation and repair of dermal, subcutaneous, and vocal cord tissue defects | |
| JPWO2002091955A1 (en) | Artificial kidney having protein metabolism function and method for producing the same | |
| DE102008008071A1 (en) | Injectable biocompatible composition | |
| JPH083199A (en) | Collagen-chondrocyte complex | |
| JP4431320B2 (en) | Transplantable medical material with self-organization promoting function | |
| Asonuma et al. | Cell transplantation as replacement therapy for the future | |
| RU2277423C1 (en) | Method for repairing structural and functional changes in connective tissue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20040316 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20040727 |