WO2002102429A1 - Biomateriaux utilises pour la reconstruction nerveuse et procede de fabrication de ceux-ci - Google Patents
Biomateriaux utilises pour la reconstruction nerveuse et procede de fabrication de ceux-ci Download PDFInfo
- Publication number
- WO2002102429A1 WO2002102429A1 PCT/JP2002/005885 JP0205885W WO02102429A1 WO 2002102429 A1 WO2002102429 A1 WO 2002102429A1 JP 0205885 W JP0205885 W JP 0205885W WO 02102429 A1 WO02102429 A1 WO 02102429A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- chitosan
- chitin
- group
- fragment
- nerve
- Prior art date
Links
Classifications
-
- 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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/04—Macromolecular materials
- A61L31/042—Polysaccharides
-
- 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
- A61L27/20—Polysaccharides
-
- 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
- A61L27/28—Materials for coating prostheses
- A61L27/34—Macromolecular materials
-
- 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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
- A61L31/10—Macromolecular materials
-
- 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
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
Definitions
- the present invention relates to a nerve-reconstructing biomaterial in which the surface of chitin or chitosan is modified with a lamien fragment, and a method for producing the same.
- PGA polyglycolic acid
- this PGA tube is the only artificial nerve used in the clinic, but there are problems with biocompatibility and affinity, and no clinical cases have been reported in Japan.
- the crosslinkable length is less than 10 cm.
- functional regeneration can be obtained by artificial nerve transplantation.
- a group at Kyoto University has developed an artificial nerve in which collagen fibers coated with laminin are inserted into a PGA collagen tube.
- ramien a glycoprotein
- a glycoprotein is flexible with a molecular weight of about 900,000. It has a simple cross-shaped molecular structure, has the function of adhering epithelial cells to connective tissue, and has the effect of promoting the elongation of neurites, which is expected to be useful for nerve reconstruction.
- laminin used in the above-mentioned conventional technology is an SHS-derived product derived from EHS rats and cannot be used in humans. In addition, it is difficult to synthesize laminin due to its high molecular weight. Disclosure of the invention
- the present inventors have made intensive research and development on a clinically applicable human nerve reconstruction biomaterial having an action of inducing regenerative nerves comparable to laminin, and by binding a laminin fragment to chitin or chitosan side chains to achieve the object. Found that it could be achieved.
- the present invention is a nerve-reconstructing biomaterial obtained by modifying a laminin fragment containing the amino acid sequence of YIGSR or IKVAV on the surface of chitin or chitosan.
- the present invention introduces a carboxyl group on the surface of chitin or chitosan, and then immerses it in a phosphate buffer containing a laminin fragment containing the amino acid sequence of YIGSR or IKVAV, and then negatively charges the carboxyl group.
- the laminin fragment is electrostatically adsorbed on the surface of chitin or chitosan by the above method.
- the present invention provides a method for binding calcium phosphate to the surface of chitin or chitosan or chitin or carboxyl-introduced chitin or chitosan into which a lipoxyl group has been introduced, and then comprises a Lamien fragment comprising the amino acid sequence of YI31 or 1 KVAV.
- a Lamien fragment comprising the amino acid sequence of YI31 or 1 KVAV.
- the present invention provides a method for introducing a carboxyl group into chitin or chitosan surface, and then immersing the carboxyl group in an aqueous solution of a revoxyl group activating reagent to activate the introduced carboxyl group.
- the carboxyl group on the surface of chitin or chitosan reacts with the hydroxyl group and amino group of the laminin fragment to form a covalent bond by immersion in a phosphate buffer containing a laminin fragment containing the sequence. It is a method of manufacturing a material.
- the present invention provides a method for introducing a lipoxyl group to the surface of chitin or chitosan, and then immersing the lipoxyl group in a lipoxyl group-activating reagent aqueous solution to activate the introduced carboxyl group, followed by a molecule having a thiol group.
- the thiol group immobilized on the carboxyl group is activated by reacting the introduced thiol group with a thiol group activating reagent to form an S--S bond, followed by YIGSR or IKVAV
- the active site of the Lamiene fragment was protected by disulfide exchange reaction between the thiol group at the terminal of the laminin fragment and the previously formed S--S bond by immersion in a phosphate buffer containing a laminin fragment containing the amino acid sequence of
- the method for producing a nerve reconstruction biomaterial as described above, which is immobilized in a state is immobilized in a state.
- the nerve-reconstructing biomaterial of the present invention as a nerve-reconstructing material, has an action of promoting elongation of nerve cells and an action of cell adhesion, and has excellent biocompatibility and biodegradability.
- FIG. 1 is a schematic diagram showing the molecular structure of laminin. BEST MODE FOR CARRYING OUT THE INVENTION
- the present invention provides a laminin fragment having an amino acid sequence such as YIGSR (tyrosine-isoleucine-glycine-serine-anoreginin) or IKVAV (isoleucine-lysine-valine-alanine-valine) on chitin or chitosan.
- YIGSR tyrosine-isoleucine-glycine-serine-anoreginin
- IKVAV isoleucine-lysine-valine-alanine-valine
- Carboxyl groups are introduced into chitin or chitosan using monochloroacetic acid or the like.
- a film or tube of chitin or chitosan is immersed in an aqueous solution of sodium hydroxide, and acetic acid or the like is added dropwise to this.
- the concentration of sodium hydroxide is 5 to 20 M. If this concentration is too high (more than 25 M), the reaction proceeds too much and chitin or chitosan is dissolved.
- Chitosan (C-chitosan) into which a lipoxyl group is introduced is soluble ⁇ fe.
- acetic acid at a monochrome mouth is added dropwise to a certain extent (for example, up to pHIO), followed by neutralization with an acid such as hydrochloric acid.
- the laminin fragment used is a Lamiyun fragment containing the amino acid sequence of YIGSR or IKVAV.
- Lamiyun fragments are electrostatically adsorbed on the surface of chitin or i-chitosan due to the negative charge of the carboxyl group.
- Bind calcium phosphate to chitin or chitosan or chitin or carboxyl group-introduced chitosan surface For example, a film or tube of chitin or chitosan is immersed in an aqueous solution of calcium chloride, calcium acetate or calcium lactate. The longer the immersion time, the greater the production of calcium-bound chitin or chitosan. Then, it is washed with saline or distilled water.
- aqueous solution containing phosphoric acid for example, an aqueous solution of sodium hydrogen phosphate, an aqueous solution of sodium dihydrogen phosphate, or an aqueous solution of diammonium hydrogen phosphate.
- This is then washed with saline or distilled water. Repeat the above series of operations several times, for example, about five times. The larger the number, the greater the amount of generation.
- This is followed by immersion in a phosphate buffer solution containing the laminin fragment.
- the laminin fragment is electrostatically adsorbed to the chitin or chitosan surface via calcium phosphate due to the negative charge of calcium phosphate.
- the activity of the modified laminant is easily maintained, but the adhesion to the surface is weaker than that of the covalent bond.
- the activated carboxyl group is activated as shown in Equation 2 below by immersing in a water-soluble carboxy group activating reagent (WSC: Water Soluble Carbodiimide. N-liydroxysccinimide) shown in (2).
- WSC Water Soluble Carbodiimide. N-liydroxysccinimide
- the WSC concentration is, for example, about 30 mM, and the immersion time is about 30 minutes. The higher the concentration of WSC, the greater the amount of modification. However, if the concentration is too high, intermolecular cross-linking in chitosan proceeds, so the introduced carboxyl groups decrease and the effect becomes worse.
- the intramolecular cross-linking between the lipoxyl group introduced into the chitosan and the hydroxyl group or amino group in the chitosan proceeds, so that the introduced carboxyl group decreases and the efficiency becomes poor.
- the immersion time is too short, the carbohydrate introduced into chitosan The xyl group may not be fully activated. Therefore, it is necessary to set an appropriate activation time.
- the temperature condition the activity is lowered at an extremely high temperature, so that the temperature is preferably about 50 ° C or less. There is no problem if the temperature is about room temperature, and it is more preferable if the temperature is about 4 ° C.
- This method has a stronger fixation force than the methods 1) and 2) above.
- OH and NH 2 are present in the active site of the laminin fragment.
- the reaction may result in loss of the activity of the lamien fragment, and the binding site for the laminin fragment cannot be specified, resulting in a decrease in the activity (for example, CDPG GY IGSR that reacts with CDPG) Keeps activity, but those that have reacted with YI GSR are inactivated).
- C-chitin or chitosan (C-chitosan) is immersed to immobilize a molecule having a thiol group such as cysteine in the carboxyl group.
- the cysteine-immobilized C-chitosan contains 5,5'-ditliiobis- (2-nitrobenzoic acid) (DTNB), which is in excess of the expected amount of cysteine immobilized.
- DTNB 5,5'-ditliiobis- (2-nitrobenzoic acid)
- C-chitin or C-chitosan on which cysteine is immobilized is immersed in a phosphate buffer, and the introduced thiol group is reacted with a thiol group activating reagent to form an S_S bond, thereby forming a lipoxyl group.
- a thiol group activating reagent to form an S_S bond, thereby forming a lipoxyl group.
- the reaction between cysteine and DTNB ends immediately after mixing, but the immersion time is about 30 minutes in consideration of the diffusion of DTNB to the fixed surface.
- This method makes the modification process complicated, but has a stronger fixation force than the above methods 1) and 2), and can specify the binding site to the laminin fragment. As a result, the activity can be maintained (reaction with C of the CDPGY I GSR maintains the activity of the YI GSR).
- a chitosan tube having a diameter of 2 mm, a length of 15 mm, and a thickness of 0.1 mm was immersed in 2 Om1 of a 10 M aqueous sodium hydroxide solution. To this, 2.5 M monochloroacetic acid was added dropwise until the pH reached 7. By this operation, lipoxyl groups were introduced on the chitosan surface.
- the obtained carboxymethylated chitosan (C-chitosan) was washed well with water to remove by-products. The confirmation of carboxylation was confirmed by detecting a carboxyl group in the analysis by FT-IR (Spectrum 2000, Perkin-Elmer).
- a chitosan tube with a diameter of 2 ⁇ , a length of 15 mm, and a thickness of 0.1 mm is immersed in a 2.2% aqueous solution of calcium chloride for 5 minutes, then taken out and immersed in physiological saline for 30 seconds. To clean the material surface. Then, the material was immersed in a 4.3% aqueous solution of disodium hydrogen phosphate for 5 minutes, then taken out, and immersed in physiological saline for 30 seconds to wash the material surface. The above series of operations was repeated five times. By performing such an operation, a calcium phosphate compound was bound to the chitosan surface.
- a chitosan tube having a diameter of 2 mm, a length of 15 mm, and a thickness of 0.1 mm was immersed in 2 Om1 of a 10 M aqueous sodium hydroxide solution. Add 2.5 M mono-acetic acid to this It was added dropwise until the pH reached 7. By this operation, a lipoxyl group was introduced into the chitosan surface ffi. The obtained carboxymethylated chitosan (C-chitosan) was thoroughly washed with water to remove by-products.
- C-chitosan carboxymethylated chitosan
- a chitosan tube having a diameter of 2 mm, a length of 15 mm, and a thickness of 0.1 ram was immersed in 2 Om 1 of a 10 M aqueous sodium hydroxide solution. To this was added dropwise 2.5 M monochloroacetic acid until the pH became 7. This operation introduced a carboxyl group on the chitosan surface. The obtained carboxymethylated chitosan (C-chitosan) was thoroughly washed with water to remove by-products.
- Cysteine immobilized C-chitosan is immersed in 1 OmM 5,5'-dithiobis- (2-nitrobenzoic acid) (DTNB) phosphate buffer (pH 8) for 30 minutes, and the cysteine is immobilized by the S—S bond formation reaction. The thiol group was activated. After that, Sigma 1668 CDPGY I GSR (laminin fragment) was immobilized at the end of the laminin fragment by diso-nolide exchange reaction by immersion in 100 g Zm1 aqueous phosphate buffer solution (pH 7.4) for 1 to 20 hours. .
- the amount of immobilized lamiyun fragment was determined by measuring the absorbance at 412 nm of 5-thio- (2-nitrobenzoic acid) (TNB) released by the disulfide exchange reaction and determining the molar extinction coefficient of TNB (E ⁇ 13, 600). As a result, it was confirmed that a laminin fragment of 19 gZ cm 2 was immobilized.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Medicinal Chemistry (AREA)
- Dermatology (AREA)
- Chemical & Material Sciences (AREA)
- Heart & Thoracic Surgery (AREA)
- Surgery (AREA)
- Vascular Medicine (AREA)
- Materials For Medical Uses (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Prostheses (AREA)
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP02733496A EP1493454A1 (en) | 2001-06-14 | 2002-06-12 | Biomaterials for nerve reconstruction and process for producing the same |
US10/480,596 US20040248777A1 (en) | 2001-06-14 | 2002-06-12 | Biomaterials for nerve reconstruction and process for producing the same |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2001180789A JP2002369877A (ja) | 2001-06-14 | 2001-06-14 | 神経再建生体材料およびその製造方法 |
JP2001-180789 | 2001-06-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2002102429A1 true WO2002102429A1 (fr) | 2002-12-27 |
Family
ID=19021150
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2002/005885 WO2002102429A1 (fr) | 2001-06-14 | 2002-06-12 | Biomateriaux utilises pour la reconstruction nerveuse et procede de fabrication de ceux-ci |
Country Status (4)
Country | Link |
---|---|
US (1) | US20040248777A1 (ja) |
EP (1) | EP1493454A1 (ja) |
JP (1) | JP2002369877A (ja) |
WO (1) | WO2002102429A1 (ja) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110091550A1 (en) * | 2009-06-04 | 2011-04-21 | Clemson University Research Foundation | Methods for Promoting the Revascularization and Reenervation of CNS Lesions |
US8609409B2 (en) | 2009-06-04 | 2013-12-17 | Clemson University | Methods and compositions for cell culture platform |
US8680182B2 (en) | 2009-06-04 | 2014-03-25 | Clemson University Research Foundation | Methods for promoting the revascularization and reenervation of CNS lesions |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4982887B2 (ja) * | 2007-02-20 | 2012-07-25 | 北海道曹達株式会社 | 神経再生チューブ及びその製造方法 |
JP5131745B2 (ja) * | 2007-09-13 | 2013-01-30 | 独立行政法人国立循環器病研究センター | 神経誘導管 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992012727A1 (en) * | 1991-01-25 | 1992-08-06 | Regents Of The University Of Minnesota | Laminin a chain domain vi polypeptides |
JP2000143531A (ja) * | 1998-09-09 | 2000-05-23 | Kuraray Co Ltd | 神経再生用材料 |
JP2000325463A (ja) * | 1999-05-18 | 2000-11-28 | Koken Co Ltd | 神経再建用基材 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NZ219198A (en) * | 1987-02-05 | 1990-11-27 | Sensasel Worldwide Ltd | Illuminated sign with proximity sensor |
US5305197A (en) * | 1992-10-30 | 1994-04-19 | Ie&E Industries, Inc. | Coupon dispensing machine with feedback |
AU7245598A (en) * | 1997-04-03 | 1998-10-22 | California Institute Of Technology | Enzyme-mediated modification of fibrin for tissue engineering |
-
2001
- 2001-06-14 JP JP2001180789A patent/JP2002369877A/ja active Pending
-
2002
- 2002-06-12 US US10/480,596 patent/US20040248777A1/en not_active Abandoned
- 2002-06-12 WO PCT/JP2002/005885 patent/WO2002102429A1/ja not_active Application Discontinuation
- 2002-06-12 EP EP02733496A patent/EP1493454A1/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992012727A1 (en) * | 1991-01-25 | 1992-08-06 | Regents Of The University Of Minnesota | Laminin a chain domain vi polypeptides |
JP2000143531A (ja) * | 1998-09-09 | 2000-05-23 | Kuraray Co Ltd | 神経再生用材料 |
JP2000325463A (ja) * | 1999-05-18 | 2000-11-28 | Koken Co Ltd | 神経再建用基材 |
Non-Patent Citations (3)
Title |
---|
222ND ACS NATIONAL MEETING (MACR-029), 26 August 2001 (2001-08-26) - 30 August 2001 (2001-08-30), CHICAGO, IL, USA * |
DATABASE CAPLUS [online] SHOICHET MOLLY S. ET AL.: "Polymers for neural tissue engineering", XP002956361, accession no. ACS Database accession no. 2001:639817 * |
TAKAKUDA KAZUO ET AL.: "A synthetic laminin peptide is active in periopheral nerve regeneration in vivo", IYO KIZAI KENKYUSHO HOKOKU (TOKYO IKA SHIKA DAIGAKU), vol. 28, 1994, pages 70 - 74, XP002956360 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110091550A1 (en) * | 2009-06-04 | 2011-04-21 | Clemson University Research Foundation | Methods for Promoting the Revascularization and Reenervation of CNS Lesions |
US8481067B2 (en) * | 2009-06-04 | 2013-07-09 | Clemson University Research Foundation | Methods for promoting the revascularization and reenervation of CNS lesions |
US8609409B2 (en) | 2009-06-04 | 2013-12-17 | Clemson University | Methods and compositions for cell culture platform |
US8680182B2 (en) | 2009-06-04 | 2014-03-25 | Clemson University Research Foundation | Methods for promoting the revascularization and reenervation of CNS lesions |
Also Published As
Publication number | Publication date |
---|---|
JP2002369877A (ja) | 2002-12-24 |
EP1493454A9 (en) | 2005-03-23 |
EP1493454A1 (en) | 2005-01-05 |
US20040248777A1 (en) | 2004-12-09 |
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