WO2013081103A1 - 耳介軟骨組織の製造方法及び耳介軟骨組織 - Google Patents
耳介軟骨組織の製造方法及び耳介軟骨組織 Download PDFInfo
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- WO2013081103A1 WO2013081103A1 PCT/JP2012/081084 JP2012081084W WO2013081103A1 WO 2013081103 A1 WO2013081103 A1 WO 2013081103A1 JP 2012081084 W JP2012081084 W JP 2012081084W WO 2013081103 A1 WO2013081103 A1 WO 2013081103A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
- A61L2/18—Liquid substances or solutions comprising solids or dissolved gases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3852—Cartilage, e.g. meniscus
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L67/00—Compositions of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Compositions of derivatives of such polymers
- C08L67/04—Polyesters derived from hydroxycarboxylic acids, e.g. lactones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
Definitions
- the present invention relates to a method for producing an auricular cartilage tissue capable of producing an auricular cartilage tissue having sufficient thickness and mechanical strength, and the auricular cartilage tissue produced by the method for producing an auricular cartilage tissue. About.
- Patent Document 1 discloses a transplant base material composed of a collagen single yarn.
- Patent Documents 2 and 3 disclose a bioabsorbable material foam, a cardiovascular tissue culture substrate reinforced with the same material, and a tube-shaped nerve regeneration substrate.
- Patent Document 4 discloses a medical material having a gel in which cells are dispersed inside a skeleton made of a sponge-like or nonwoven-like polymer material molding.
- the auricular cartilage tissue is required to have a relatively high mechanical strength in addition to a large and thick tissue.
- the present invention relates to a method for producing an auricular cartilage tissue capable of producing an auricular cartilage tissue having sufficient thickness and mechanical strength, and the auricular cartilage tissue produced by the method for producing an auricular cartilage tissue.
- the purpose is to provide.
- the present invention provides a cell seeding step of seeding auricular chondrocytes on a nonwoven fabric made of a bioabsorbable material having an average fiber diameter of 0.90 to 7.00 ⁇ m, and a nonwoven fabric seeded with the auricular chondrocytes. It is a method for producing an auricular cartilage tissue having a forming step of adjusting a shape by combining with a mesh-shaped mold made of a non-absorbable material. The present invention is described in detail below.
- the present inventors tried to regenerate auricular cartilage tissue by seeding auricular chondrocytes on a support having various materials and shapes.
- a non-woven fabric made of a bioabsorbable material having an average fiber diameter of 0.90 to 7.00 ⁇ m is used, the auricular cartilage is remarkably compared to the case of using a support having another material or shape.
- the regeneration of the organization is promoted.
- after seeding the auricular chondrocytes on a non-woven fabric made of a bioabsorbable material having an average fiber diameter of 0.90 to 7.00 ⁇ m this was formed into a mesh-shaped formwork made of a non-bioabsorbable material.
- the inventors have found that an auricular cartilage tissue having sufficient thickness and mechanical strength can be produced by compounding and molding, and the present invention has been completed.
- the method for producing auricular cartilage tissue of the present invention seeds auricular chondrocytes on a nonwoven fabric (hereinafter also simply referred to as “nonwoven fabric”) made of a bioabsorbable material having an average fiber diameter of 0.90 to 7.00 ⁇ m. Cell seeding step.
- a nonwoven fabric composed of such a specific average fiber diameter and a specific material, an auricular cartilage tissue having a sufficient thickness and mechanical strength can be produced.
- the bioabsorbable material is not particularly limited.
- polyglycolide, polylactide (D, L, DL form), polycaprolactone, glycolic acid-lactide (D, L, DL form) copolymer, glycolic acid- ⁇ - Examples include caprolactone copolymer, lactide (D, L, DL form) - ⁇ -caprolactone copolymer, poly (p-dioxanone) and the like. These may be used independently and may use 2 or more types together. Among these, polyglycolide or lactide (D, L, DL form) - ⁇ -caprolactone copolymer is preferable, and polyglycolide is more preferable.
- the nonwoven fabric has an average fiber diameter of 0.90 to 7.00 ⁇ m.
- the average fiber diameter of the nonwoven fabric is within this range, regeneration of the auricular cartilage tissue is promoted.
- the average fiber diameter of the nonwoven fabric is 0.90 ⁇ m, the regeneration of the auricular cartilage tissue is particularly promoted.
- the average fiber diameter of the nonwoven fabric is obtained by cutting out a part of the center of the nonwoven fabric, observing it using an electron microscope, and randomly extracting the focused fibers, It means a value obtained by averaging the diameters of the obtained 100 or more fibers when the diameter of the fibers is measured while changing the place until it becomes 100 or more.
- the said nonwoven fabric has a preferable minimum of a fabric weight of 1 g / m ⁇ 2 >, and a preferable upper limit of 100 g / m ⁇ 2 >. If it is out of this range, the auricular cartilage tissue may not be sufficiently regenerated.
- the more preferable lower limit of the basis weight of the nonwoven fabric is 5 g / m 2
- the more preferable upper limit is 50 g / m 2 .
- a method for producing a nonwoven fabric made of a bioabsorbable material having an average fiber diameter of 0.90 to 7.00 ⁇ m is not particularly limited.
- electrospinning deposition method melt blow method, needle punch method, spun bond method, flash Conventionally known methods such as spinning method, hydroentanglement method, airlaid method, thermal bond method, resin bond method, and wet method can be used.
- the melt blow method is preferable.
- auricular chondrocytes are seeded on the nonwoven fabric.
- the auricular chondrocytes can be collected by a conventionally known method. For example, after removing skin, connective tissue, and perichondrium from an auricle obtained from a human or an animal, etc., the auricular chondrocytes are isolated by cutting into small pieces of about 5 mm ⁇ 5 mm and then treating with collagenase. Can be separated.
- the isolated auricular chondrocytes may be directly used for the method for producing auricular cartilage tissue of the present invention, or may be used for the method for producing auricular cartilage tissue of the present invention after being grown by culture.
- the seeding method is not particularly limited, and a conventionally known seeding method can be used.
- the seeding density at the time of sowing is not particularly limited, but a preferable lower limit is 2.0 ⁇ 10 7 cells / cm 2 and a preferable upper limit is 1.0 ⁇ 10 8 cells / cm 2 .
- a preferable lower limit is 2.0 ⁇ 10 7 cells / cm 2
- a preferable upper limit is 1.0 ⁇ 10 8 cells / cm 2 .
- the cell seeding density is less than 2.0 ⁇ 10 7 cells / cm 2 , it may take time until an auricular cartilage tissue having sufficient thickness and mechanical strength is formed. Even if cells are seeded in excess of 10 8 cells / cm 2 , no further effect is observed.
- a more preferable lower limit of the cell seeding density is 5.0 ⁇ 10 7 cells / cm 2 .
- the non-woven fabric seeded with the auricular chondrocytes is preferably allowed to stand for about 10 minutes until the auricular chondrocytes adhere sufficiently. Moreover, you may culture
- a culture solution for culturing for example, a serum-added medium in which about 1 to 10% by weight of fetal bovine serum is added to a general culture solution such as MEM or DMEM can be used.
- the method for producing auricular cartilage tissue according to the present invention includes a molding step in which the nonwoven fabric seeded with the auricular chondrocytes is combined with a mesh-shaped mold made of a non-bioabsorbable material to adjust the shape.
- a mesh-shaped mold made of a non-bioabsorbable material In order to regenerate the auricular cartilage tissue, it is necessary to form a large and thick tissue. It is also important to arrange the shape according to the transplant site.
- the mesh-shaped formwork made of the non-bioabsorbable material plays a role of adjusting the obtained auricular cartilage tissue to an arbitrary shape and an arbitrary thickness.
- the non-bioabsorbable material forming the mesh-shaped form is not particularly limited as long as it is not toxic to the living body and has appropriate hardness and elasticity.
- polypropylene, polyethylene, polytetrafluoroethylene (PTEF) ), Nylon and the like are preferred.
- the mesh-shaped formwork preferably has a shape corresponding to the auricular cartilage tissue to be regenerated.
- a shape corresponding to the entire auricle it is preferable to have a shape corresponding to the entire auricle.
- the entire auricle may be divided into a plurality of parts and combined into a shape corresponding to each of the parts to form the entire auricle.
- the method of combining the nonwoven fabric seeded with the auricular chondrocytes and the mesh mold is not particularly limited, and the nonwoven fabric seeded with the auricular chondrocytes is sandwiched between two mesh molds. It may be molded into an arbitrary shape, or a non-woven fabric seeded with auricular chondrocytes may be wrapped so as to wrap a mesh-shaped formwork of an arbitrary shape.
- the auricular cartilage tissue produced by the method for producing auricular cartilage tissue of the present invention By transplanting the auricular cartilage tissue produced by the method for producing auricular cartilage tissue of the present invention into a living body, the auricular cartilage tissue having sufficient thickness and mechanical strength is regenerated.
- An auricular cartilage tissue composed of a non-woven fabric made of a bioabsorbable material having an average fiber diameter of 0.90 to 7.00 ⁇ m seeded with auricular chondrocytes and a mesh-like formwork made of a non-bioabsorbable material Moreover, it is one of the present inventions.
- an auricular cartilage tissue manufacturing method capable of manufacturing an auricular cartilage tissue having sufficient thickness and mechanical strength, and an auricle manufactured by the auricular cartilage tissue manufacturing method.
- Cartilage tissue can be provided.
- Example 1 Preparation of non-woven fabric
- the average fiber diameter is 0.67 ⁇ m (sample I), 0.90 ⁇ m by a method in which a non-woven fabric obtained by a melt-blowing method is drawn or spun into a non-woven fabric by a needle punch method.
- Sample II A nonwoven fabric having a thickness of 0.13 to 0.30 mm made of polyglycolide of 3.10 ⁇ m (sample III), 7.00 ⁇ m (sample IV), and 20.60 ⁇ m (sample V) was obtained. .
- FIG. 1 the bar below the photograph indicates 30 ⁇ m.
- the focused fibers in the electron micrograph were randomly extracted and the diameter of each fiber was measured. The diameter of the fiber was measured while changing the location until the total of the measurement reached 100, and the diameter of the 100 obtained fibers was averaged to obtain an average fiber diameter of 3.10 ⁇ m. For other samples I, II, IV and V, the average fiber diameter was obtained in the same manner.
- a sheet-like mesh having a size of 2 cm ⁇ 2 cm and a thickness of 0.34 ⁇ 0.007 mm made of a composite polypropylene with a mesh-shaped mold was prepared.
- a non-woven fabric seeded with auricular chondrocytes was sandwiched between two sheet-like meshes to form a flat plate-like scaffold.
- a three-dimensional shape maintenance confirmation sample a non-woven fabric seeded with auricular chondrocytes was formed into a pinna shape by sandwiching a non-woven fabric seeded with a seed-like mesh to obtain a human pinna shaped scaffold.
- FIG. 1 shows a photograph of the appearance of a sample for confirmation of maintenance of the three-dimensional shape taken out and a stained photograph of the cross section.
- the cross section of the sample is cut by the line portion in the upper diagram of FIG. 1 and then stained by the safranin O staining method.
- a nonwoven fabric (samples II to V) having an average fiber diameter of 0.90 ⁇ m or more is used, the regenerated cartilage is sufficiently induced, whereas the average fiber diameter is 0.67 ⁇ m (sample I). It can be seen that the regenerated cartilage was insufficiently induced when the non-woven fabric of (1) was used.
- the average fiber diameter was 0.67 ⁇ m (Sample I), 0.90 ⁇ m (Sample II), 3.10 ⁇ m (Sample III), 7.00 ⁇ m (Sample IV), and 20.60 ⁇ m (by the same method as in the experimental example)
- a non-woven fabric having a thickness of 0.13 to 0.30 mm made of polyglycolide of sample V) was obtained.
- Both pinna of beagle dogs (female, 6-8 weeks old) were cut under anesthesia.
- the obtained small piece was first treated in a collagenase solution with a concentration of 0.3%, and then the auricular chondrocytes were isolated.
- the isolated auricular chondrocytes were suspended in a phosphate buffer to obtain a cell suspension of 1.0 ⁇ 10 8 cells / mL. 200 ⁇ L of the obtained cell suspension was seeded on a nonwoven fabric cut to a size of 2 cm ⁇ 2 cm.
- the non-woven fabric after sowing was divided into two groups, and fibrin glue (mixed solution of vibrinogen and thrombin) was sprayed on one group. After spraying fibrin glue, the mixture was allowed to stand for 5 minutes, and then both groups were immersed in a 2.5% glutaraldehyde solution to obtain a sample. After cutting out every 100 ⁇ m of the nonwoven fabric and staining with toluidine blue, the chondrocytes infiltrating into the nonwoven fabric were counted and the number of cells was counted. The results are shown in Table 2.
- the average fiber diameter is 0.67 ⁇ m (sample I)
- the cell density is low, there is a space in the middle, and there are not enough cells.
- the average fiber diameter was 20.60 ⁇ m (sample V)
- the cell density was low, and the cells were distributed only around the fiber bundle, which was not uniform.
- the average fiber diameter was 0.90 ⁇ m (Sample II) to 7.00 ⁇ m (Sample IV)
- a cell having a high cell density was obtained.
- an auricular cartilage tissue manufacturing method capable of manufacturing an auricular cartilage tissue having sufficient thickness and mechanical strength, and an auricle manufactured by the auricular cartilage tissue manufacturing method.
- Cartilage tissue can be provided.
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Abstract
Description
また、特許文献2及び特許文献3には、生体吸収性素材の発泡体と、同様素材により補強した心血管系組織培養基材、並びにチューブ状の神経再生基材が開示されている。
更に、特許文献4には、スポンジ状または、不織布状の高分子材料成形物からなる骨格の内部に細胞を分散したゲルを有する医用材料が開示されている。
以下に本発明を詳述する。
なお、本明細書において不織布の平均繊維径は、不織布の生地の中央の一部を切り取り、電子顕微鏡を用いて観察し、焦点が合っている繊維を無作為に抽出して、測定の合計が100本以上となるまで場所を変えながら繊維の直径を測定したときに、得られた100本以上の繊維の直径を平均した値を意味する。
上記耳介軟骨細胞は、従来公知の方法により採取することができる。例えば、人や動物等から得た耳介から、皮膚、結合織、軟骨膜を除去したうえで、5mm×5mm程度の小片に細切した後、コラゲナーゼ処理することにより、耳介軟骨細胞を単離することができる。単離した耳介軟骨細胞は、そのまま本発明の耳介軟骨組織の製造方法に供してもよく、培養により増殖させた後に本発明の耳介軟骨組織の製造方法に供してもよい。
上記播種の際の播種の密度は特に限定されないが、好ましい下限は2.0×107cells/cm2、好ましい上限は1.0×108cells/cm2である。細胞播種密度が2.0×107cells/cm2未満であると、充分な厚みと力学的強度とを有する耳介軟骨組織が形成されるまでに時間がかかることがあり、1.0×108cells/cm2を超えて細胞を播種しても、それ以上の効果は認められない。細胞播種密度のより好ましい下限は5.0×107cells/cm2である。
耳介軟骨組織の再生には、大型で厚みのある組織が形成されることが必要である。また、移植部位に合わせた形状を整えることも重要である。上記生体非吸収性材料からなるメッシュ状の型枠は、得られる耳介軟骨組織を任意の形状、任意の厚みに調整する役割を果たすものである。
耳介軟骨細胞が播種された、平均繊維径が0.90~7.00μmの生体吸収性材料からなる不織布と、生体非吸収性材料からなるメッシュ状の型枠とからなる耳介軟骨組織もまた、本発明の1つである。
(1)不織布の調製
メルトブロー法で得られた不織布を延伸、又は、紡糸された筒編み布をニードルパンチ法により不織布化する方法により、平均繊維径が0.67μm(サンプルI)、0.90μm(サンプルII)、3.10μm(サンプルIII)、7.00μm(サンプルIV)、及び、20.60μm(サンプルV)のポリグリコリドからなる、厚さ0.13~0.30mmの不織布を得た。
図1に示したように、電子顕微鏡写真中の焦点が合っている繊維を無作為に抽出し、各々の繊維の直径を測定した。測定の合計が100本となるまで場所を変えながら繊維の直径を測定し、得られた100本の繊維の直径を平均して、3.10μmという平均繊維径を得た。他のサンプルI、II、IV、Vについても同様の方法により平均繊維径を得た。
ビーグル犬(メス、6~8週齢)の両耳介を、麻酔下で切断した。得られた耳介から、皮膚、結合織、軟骨膜を除去したうえで、5mm×5mm程度の小片に細切した。得られた小片を、濃度0.3%のコラゲナーゼ溶液中で1晩処理した後、耳介軟骨細胞を単離した。単離した耳介軟骨細胞をリン酸バッファー中に懸濁させて、1.0×108cells/mLの細胞懸濁液を得た。
ポリプロピレンからなる大きさが2cm×2cm、厚さが0.34±0.007mmのシート状のメッシュを準備した。
曲げ強度測定用サンプルとして、耳介軟骨細胞が播種された不織布を2枚のシート状メッシュに挟んで複合化し、平板型のスカフォールドを作製した。
一方、3次元形状維持確認用サンプルとして、耳介軟骨細胞が播種された不織布を、シード状メッシュにて挟んだパーツを耳介形状に成形し、ヒト耳介形状のスカフォールドを得た。
得られた平板型のスカフォールド及びヒト耳介形状のスカフォールドを同一の個体(ビーグル犬、メス、6~8週齢)に自家移植した。全身麻酔後、後頸部に切開し、頭部筋膜間にスカフォールドを移植、固定した。移植後5週間後に犠牲死させて、各々のサンプルを取り出した。
(1)曲げ強度の測定
取り出した曲げ強度測定用サンプルについて、ROYらの方法に従い、オートグラフを用いて曲げ強度を測定した。即ち、グリップ間を1cmに調整した後、20mm×5mmの大きさにしたサンプルを台座に固定し、垂直板を0.02mm/secの速度で下降させる条件により曲げ強度を測定した。
結果を表1に示した。
取り出した3次元形状維持確認用サンプルの外見の写真と、断面部の染色写真を図1に示した。サンプルの断面は、図1上図の線部分で切断した後、サフラニンO染色法により染色を行ったものである。
図1より、平均繊維径が0.90μm以上の不織布(サンプルII~V)を用いた場合には、再生軟骨の誘導が充分であるのに対して、平均繊維径が0.67μm(サンプルI)の不織布を用いた場合には、再生軟骨の誘導が不充分であったことが判る。
実験例と同様の方法により、平均繊維径が0.67μm(サンプルI)、0.90μm(サンプルII)、3.10μm(サンプルIII)、7.00μm(サンプルIV)、及び、20.60μm(サンプルV)のポリグリコリドからなる、厚さ0.13~0.30mmの不織布の不織布を得た。
得られた細胞懸濁液200μLを、2cm×2cmの大きさに切断した不織布上に播種した。
フィブリン糊散布後、5分間静置した後、両群を2.5%グルタルアルデヒド溶液中に浸漬してサンプルを得た。
不織布100μm毎に切り出しを行い、トルイジンブルーにて染色を行った後、不織布内部に浸潤している軟骨細胞の計測を行い、細胞数を計数した。
結果を表2に示した。
よって、軟骨再生組織に利用する不織布の平均繊維径が0.90μm(サンプルII)~7.00μm(サンプルIV)のものが、耳介軟骨の再生には最適である。
Claims (4)
- 平均繊維径が0.90~7.00μmの生体吸収性材料からなる不織布上に耳介軟骨細胞を播種する細胞播種工程と、
前記耳介軟骨細胞が播種された不織布を、生体非吸収性材料からなるメッシュ状の型枠と複合化して形状を整える成形工程とを有する
ことを特徴とする耳介軟骨組織の製造方法。 - 生体吸収性材料からなる不織布の平均繊維径が0.90μmであることを特徴とする請求項1記載の耳介軟骨組織の製造方法。
- 生体吸収性材料がポリグリコリド又はラクチド(D、L、DL体)-ε-カプロラクトン共重合体であることを特徴とする請求項1又は2記載の耳介軟骨組織の製造方法。
- 耳介軟骨細胞が播種された、平均繊維径が0.90~7.00μmの生体吸収性材料からなる不織布と、生体非吸収性材料からなるメッシュ状の型枠とからなる耳介軟骨組織。
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