JP5131745B2 - Nerve guide tube - Google Patents

Description

  The present invention relates to a nerve guide tube. More specifically, the present invention relates to a nerve guide tube characterized by comprising as a main component an oligolactic acid modified with a peptide and a biodegradable polymer, which can regenerate a deficient nerve tissue more efficiently.

  Deficient peripheral nerve tissue was made by surgically stitching the damaged ends together. However, when the gap between damaged ends is longer than a certain length, it is known that tension is generated when the ends are stitched, and the regeneration process is hindered by stagnant blood flow. In many cases, the stumps of the defective peripheral nerve tissue are damaged, and it is known that if such damaged stumps are sutured as they are, the damaged stumps become scarred and nerve regeneration is inhibited. . Therefore, when the stump of the deficient peripheral nerve tissue is damaged, it is necessary to excise the stump, which makes it difficult to suture the end. If end-to-end suture is difficult, autologous nerve transplantation is performed. In autologous nerve transplantation, sensory nerves that can tolerate functional loss are used so as not to impair motor function. But even sensory nerves have the potential to cause sensory impairments, and there is nothing unnecessary. In addition, it can be easily imagined that it is not a good idea to put a scalpel in the living body more than necessary. Therefore, in order to avoid autologous nerve transplantation, development of a nerve guide tube (guide tube) is required.

  So far, various studies have been made on a method for forming a nerve cell growth scaffold using an artificial instrument, regenerating nerves, and restoring the original function. However, when only the tubular body was used, some cell growth was seen from both ends of the cut nerve, but it was difficult for the cut nerve to rejoin and recover. This is because when cells proliferate, they generally adhere to the scaffold of the tubular body and then grow in the direction of filling the cut part, but there is a gap between the cut ends just by covering the cut part, This is because the cell growth stops until all the parts are filled.

  Silicon was first considered as a guide tube made of artificial materials. Silicon tubes have relatively few foreign body reactions in vivo, and have been successful not only in animal experiments but also in humans. However, there was no biocompatibility and no axonal extension along the inner wall of the tube was observed. Moreover, body fluid cannot be exchanged between the inside and the outside of the tube. Furthermore, it is not bioabsorbable, and discomfort and pain are caused by long-term implantation. Therefore, there was a problem that a second operation for removing the tube after nerve regeneration was necessary.

  As a more ideal guide tube, attempt to regenerate nerves using a tubular body of bioabsorbable material that does not remain in the body so that a second surgery to remove the tube is not necessary (See Non-Patent Document 1).

  Further, as a bioabsorbable material, synthetic polymer polyglycolic acid (PGA), PLA, natural polymer collagen, and the like were studied. However, there is a problem that the synthetic polymer material is slow to regenerate and a natural polymer material has a problem that the strength is insufficient. Furthermore, in order to compensate for these, composite materials of PGA, collagen, and laminin have been developed (see Non-Patent Document 2). However, collagen and laminin are not completely removed. On the other hand, in a hybrid polylactic acid-caprolactone copolymer (P (LA / CL)) tube in which Schwann cells, which are said to have a great influence on nerve regeneration, are cultured in advance in the tube, good results have been obtained. (Refer nonpatent literature 3).

Furthermore, various attempts have been made to regenerate nerves by forming a scaffold for inducing proliferation of nerve cells inside the tubular body of bioabsorbable material. For example, there is one in which a fiber bundle of collagen is inserted into a tubular body and coated with fibronectin (FN) (see Patent Document 1 and Non-Patent Document 4).
Kyoko Suzuki et al., “Artificial Organs”, 1998, 27, No. 2, p. 490-494 Matsumoto K, Ohnishi K, Kiyotani T, Sekin T, Ueda H, Nakamura T, Endo K, Shimizu Y, “Brain Research”, June 2000, 868, p. 315-328 Hadlock T, Elisseeff J, Langer R. Vacanti J, Cheney M, “Arch Otalyngol Head Neck Surg”, 1998, Vol. 124, p. 1081-1086 Shimada Hiroki et al., “Artificial Organ”, 1993, Vol. 22, No. 2, p. 359-363 JP-A-5-237139

  As described above, a collagen fiber bundle inserted into a tubular body and coated with fibronectin (FN) (see Patent Document 1 and Non-Patent Document 4) has insufficient strength, so the end of the tube is There was a risk of tearing easily at the stitched portion.

  In addition, as described above, good results have been obtained with a hybrid polylactic acid-caprolactone copolymer (P (LA / CL)) tube (see Non-Patent Document 3), but it takes time to culture autologous Schwann cells. There was a problem that it was not possible to deal with in an emergency.

  Accordingly, an object of the present invention is to provide a nerve guide tube having sufficient strength that is bioabsorbable, safe and can be used immediately in an emergency.

  As a result of intensive studies to solve the above problems, the present inventors have invented a nerve induction tube using oligolactic acid modified with a peptide.

  That is, the first gist of the present invention resides in a nerve guide tube formed by molding a resin composition containing a compound in which a peptide and oligolactic acid are bonded by a peptide bond. According to a second aspect of the present invention, there is provided a nerve guide tube according to the first aspect of the present invention, wherein the peptide has at least the Ile-Lys-Val-Ala-Val sequence. Exist. The third gist of the present invention is the nerve induction tube according to the first or second gist of the present invention, wherein the oligolactic acid is represented by the following general formula 1 (m represents an integer of 1 to 100). It exists in a nerve guide tube characterized by being expressed.

  Furthermore, the fourth gist of the present invention is the nerve guide tube according to the first to third gist of the present invention, wherein the resin composition contains a biodegradable polymer. Exist. According to a fifth aspect of the present invention, there is provided a nerve guide tube according to any one of the first to fourth aspects of the present invention, wherein cells grow along the inner wall of the nerve guide tube. Exist. Furthermore, the sixth gist of the present invention is a peptide-modified oligolactic acid (n is characterized by comprising an oligolactic acid unit represented by the following general formula 2 and a peptide represented by the following general formula 3. An integer of 1 to 500, o represents an integer of 1 to 10. A and B each represents a single bond or a divalent organic group.

  The seventh gist of the present invention is a molded article formed by molding a resin composition containing oligolactic acid modified with a peptide according to the sixth gist of the present invention. In the nerve guide tube according to the first, fourth, or fifth aspect of the present invention, the peptide represented by the general formula 3 and the oligolactic acid having the oligolactic acid unit represented by the general formula 2 are bound by a peptide bond. It exists in the nerve guide tube characterized by shape | molding the resin composition containing a compound.

  According to the present invention, it is possible to provide a nerve guide tube that is absorbed in vivo and has a sufficient strength and is effective for nerve growth.

  The lactic acid of the oligolactic acid in the present invention is a lactic acid selected from L-lactic acid, D-lactic acid, and a mixture of L-lactic acid and D-lactic acid at an arbitrary ratio, and the oligolactic acid means an oligomer of lactic acid. . The term “oligomer” means a compound having a structure in which a plurality of monomers such as lactic acid are bonded. In the case of the oligolactic acid of the present invention, the number n of lactic acid monomers is usually 2 or more and 500 or less, and the lower limit of n is preferably 3, more preferably 5, and even more preferably 10. . The upper limit of n is preferably 100, more preferably 50, and even more preferably 25. When n is larger than 500, the effect of the peptide is reduced.

  In addition, when the oligolactic acid modified with the peptide of the present invention is used in a nerve induction tube, the number m of lactic acid monomers is usually 2 or more and 100 or less, but the lower limit of m is preferably 3, more preferably 5, even more preferably 10. The upper limit of m is preferably 50, more preferably 30, and further preferably 25. When m is larger than 100, oligolactic acid modified with a peptide is difficult to migrate to the surface of the nerve guide tube, which is not preferable.

  The oligolactic acid modified with a peptide means a compound in which oligolactic acid and a plurality of amino acids are bonded by peptide bonds, and the amino acid is usually an α-amino acid. Examples of the α-amino acid include L-isoleucine, D-isoleucine, L-alloisoleucine, D-alloisoleucine, L-lysine, D-lysine, L-valine, D-valine, L-α-alanine, D-α. -Alanine, glycine, L-norvaline, D-norvaline, L-leucine, D-leucine, L-norleucine, D-norleucine, L-phenylalanine, D-phenylalanine, L-tyrosine, D-tyrosine, etc. Among these, L-isoleucine, D-isoleucine, L-alloisoleucine, D-alloisoleucine, L-lysine, D-lysine, L-valine, D-valine, L-α-alanine, D-α-alanine are preferable. Used.

  The peptide part contained in oligolactic acid modified with a peptide shows a structure in which a plurality of amino acid sequences are linked. The number of amino acid sequence bonds is usually 1 or more and 50 or less. The lower limit of the number of amino acid bonds is preferably 2, more preferably 3, and the upper limit is preferably 10, more preferably 7. The most preferred value of the number of amino acid bonds is 5.

  In the present invention, the most preferred structure of the peptide moiety contained in oligolactic acid modified with a peptide is represented by the following general formula 3.

  In the said General formula 3, o represents a positive integer, Preferably it is 1-10. It is thought that the effect of becoming easier to contact with nerve cells is obtained when o is larger. However, if o becomes too large with respect to m, the water solubility becomes high, and there is a possibility that it may not be mixed well when it is mixed with polylactic acid, so that it may elute, so the hydrophilic / hydrophobic balance with oligolactic acid must be considered. I must.

  In the above general formula 3, A and B each represent an organic group such as a peptide, a divalent aromatic group, a divalent alkyl group, an alkenyl group, oxygen, sulfur, ester, ether, urethane, etc. Preferable is a peptide or a single bond, and most preferably A and B are both a single bond.

  Examples of the method for synthesizing the oligolactic acid of the present invention include a method of synthesizing a plurality of lactic acids by dehydration polycondensation reaction, a method of synthesizing by transesterification of lactic acid ester, a method of synthesizing by lactide ring-opening reaction, A method of synthesizing by a dehydration polycondensation reaction of a plurality of lactic acids is preferably selected.

  When synthesizing oligolactic acid by dehydration polycondensation reaction of plural lactic acids, it is preferable to inactivate the hydroxyl group of oligolactic acid after synthesizing oligolactic acid and before synthesizing oligolactic acid modified with peptide. As a method of inactivation, a method of acetylation with acetic anhydride or the like is preferably used.

  Peptide-modified oligolactic acid can be synthesized by reacting terminal acetylated oligolactic acid with a peptide quantifier, or by binding amino acids to terminal acetylated oligolactic acid to extend the peptide chain. However, it is preferable to react the terminal acetylated oligolactic acid with a peptide quantifier.

  The biodegradable polymer in the present invention refers to a polymer that is degraded by microorganisms or a polymer that is degraded in vivo. For example, poly L-lactic acid, poly D-lactic acid, poly DL-lactic acid, polylactic acid-polyether Polymers, polylactic acid glycolic acid copolymers, polyglycolic acid, polycaprolactone, polybutylene succinate, polyethylene succinate, polybutylene succinate adipate, polypropylene carbonate, polyparadioxanone, and other synthetic polymers, polysaccharides, Examples thereof include natural polymers such as collagen and gelatin, and microbial polymers such as poly-4-hydroxybutyrate. Among these, poly L-lactic acid, poly D-lactic acid, poly DL-lactic acid, polyglycolic acid, polylactic acid glycolic acid copolymer, and polylactic acid-polyether copolymer are preferable, and poly L-lactic acid, D-lactic acid, polylactic acid-polyether copolymer, more preferably poly-L-lactic acid and poly-D-lactic acid, and most preferably poly-L-lactic acid.

  The ratio of the total amount of oligolactic acid modified with peptide and biodegradable polymer in the material constituting the nerve guide tube of the present invention is 50% by mass or more, preferably 70% by mass or more, more preferably Is 80% by mass or more, and most preferably 90% by mass or more. Of the materials constituting the nerve guide tube, materials other than oligolactic acid modified with peptides and biodegradable polymers may contain plasticizers, stabilizers, bioabsorbable regulators, surface treatment agents, and the like. However, a plasticizer is preferably added. Examples of the plasticizer include polyether and polyester. Examples of the polyether include polyethylene glycol, polypropylene glycol, and polyethylene glycol / polypropylene glycol copolymer, and examples of the polyester include polycaprolactone. When the ratio of the total amount of oligolactic acid modified with peptide and biodegradable polymer in the material constituting the nerve guide tube of the present invention is less than 50% by mass, the mechanical strength is reduced, or the nerve The growth promoting action is impaired, which is not preferable.

  Instead of adding polyether as a plasticizer to polylactic acid, one or more components selected from glycolic acid, polyether, and caprolactone may be copolymerized. Of these, polylactic acid-polyether copolymers and polylactic acid-polyglycolic acid are preferred, and polylactic acid-polyethylene glycol block copolymers are more preferred. The lactic acid unit contained in the copolymer when copolymerized is preferably 30% by mass or more, more preferably 50% by mass or more, and most preferably 70% by mass or more.

  The mass ratio of the oligolactic acid modified with the peptide constituting the nerve guide tube of the present invention and the biodegradable polymer is usually 10 parts by mass or less for the oligolactic acid modified with the peptide with respect to 100 parts by mass of the biodegradable polymer. is there. The upper limit of oligolactic acid modified with a peptide is preferably 40 parts by mass, more preferably 10 parts by mass, and even more preferably 5 parts by mass. The lower limit of the oligolactic acid modified with a peptide is preferably 0.01 parts by mass, more preferably 0.1 parts by mass, and even more preferably 1 part by mass. When the amount of oligolactic acid modified with a peptide is more than 40 parts by mass, the strength of the nerve guide tube is lowered, which is not preferable. On the other hand, when the amount of oligolactic acid modified with a peptide is less than 0.01 parts by mass, the effect of promoting the growth of nerve cells decreases, which is not preferable.

  The nerve guide tube of the present invention can be prepared by a known molding method. Examples of molding methods include injection molding, cast molding, spin coating, and electrospinning. Cast molding, spin coating, and electrospinning are preferable because they can be molded at low temperatures. In order to form a phase separation structure, spin coating and electrospinning are preferably used. Electrospinning is most preferable from the viewpoint of production efficiency.

  The nerve guide tube of the present invention may be subjected to physical or chemical surface treatment or crosslinking after being molded into a target shape, or may be molded into a target shape after surface treatment or crosslinking. Two or more kinds of surface treatments or crosslinking may be used in combination.

  The nerve guide tube of the present invention is preferably sterilized by a known method such as γ-ray sterilization or ultraviolet sterilization before use.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples as long as the gist thereof is not exceeded.

Example 1
(1) IKVAV peptide synthesis Each oligopeptide was synthesized by Fmoc solid-phase synthesis and manually synthesized in the air. In order to synthesize the pentameric oligopeptide IKVAV, first, Fmoc-PAL-PEG-PS (9-fluorenylmethoxycarbonyl-PAL-polyethylene glycol-polystyrene) resin (hereinafter simply referred to as “resin”) is used. Weighed into an empty plastic column and swollen with dimethylformamide (DMF). Thereafter, Fmoc-amino acid is continuously added to the resin in the order of Fmoc-amino acid (V), Fmoc-amino acid (A), Fmoc-amino acid (V), Fmoc-amino acid (K), and Fmoc-amino acid (I). Condensed. Specifically, Fmoc-amino acid was used in an amount equivalent to 3 times the amount of the above resin, and 20% piperidine / DMF was used for removal of Fmoc (protecting group) (de-Fmoc). As the activator, 3-fold equivalent of N-hydroxybenzotriazole monohydrate (HOBt) and 3-fold equivalent of 2- (1H-benzotriazole-1,1,3,3) with respect to the resin. -Tetramethyluronium hexafluorophosphate (HBTU) and 6 equivalents (1.50 mmol) of diisopropylethylamine (DIPEA) were used.

  Whether or not de-Fmoc and coupling were performed in the synthesis process of the oligopeptide IKVAV was confirmed by Kaiser test for each reaction. In de-Fmoc, the reaction is repeated until a Kaiser test is performed and negative (yellow) is positive (blue), and in a coupling, a Kaiser test is performed and positive (blue) is negative (yellow) The reaction was repeated. In this way, oligopeptide IKVAV was attached to the resin.

  A part of the obtained resin with oligopeptide IKVAV was collected, washed with DMF, DCM and methanol in this order, dried under reduced pressure, and then 85% trifluoroacetic acid (TFA) and 7.5% m as a scavenger. -A cocktail solution was prepared with cresol and 7.5% thioanisole, which was added to each column and allowed to react for approximately 1 hour at room temperature. Thereafter, the reaction solution obtained in this reaction was taken out into a centrifuge tube, and the resin after the reaction was washed 5 times with TFA, and the washing solution was also taken into the centrifuge tube. Then, after volatilizing until the volume of TFA became 1/10, it was dropped into about 20 times equivalent of diethyl ether and reprecipitated. Thereafter, the supernatant was removed by cooling and centrifugation (8500 rpm, 0 ° C., 20 minutes). This reprecipitation was repeated 3 to 6 times and dried under reduced pressure to obtain a crude oligopeptide. In this way, oligopeptide IKVAV was excised from the resin.

  The obtained crude oligopeptide was monitored by HPLC at 220 nm (absorption wavelength of peptide bond) and 300 nm (absorption wavelength of Fmoc group) for analysis. As a result, it was confirmed that a main peak was observed at 220 nm and no absorption was observed at 300 nm.

(2) Oligolactic acid synthesis Oligolactic acid was synthesized by dehydrating polycondensation reaction of lactic acid monomers. Specifically, a 90% aqueous solution of lactic acid was heated at 145 ° C. for 3 hours, then reduced in pressure at 145 ° C., and held at about 20 kPa (150 Torr) for 3 hours. Thereafter, a dehydration condensation reaction was carried out at 155 ° C. and about 0.4 kPa (3 Torr) for 3 hours, and at 185 ° C. and about 0.4 kPa (3 Torr) for 3 hours. After synthesizing oligolactic acid in this way, the hydroxyl group of the synthesized oligolactic acid was inactivated with acetic anhydride to obtain oligolactic acid (acOLLA) whose terminal was acetylated.

(3) Synthesis of acOLLA-IKVAV conjugate A part of the resin with oligopeptide IKVAV synthesized in (1) was collected. Then, without cutting out oligopeptide IKVAV from the resin, as an activator, 5 times equivalent HOBt, 5 times equivalent HBTU, and 10 times equivalent DIPEA were added to the resin as a solvent. DMF was added, and 5 times the same amount of acOLLA obtained in (2) was added to carry out coupling on the solid phase. Whether or not the coupling was performed was confirmed from the negative (yellow) in the Kaiser test as in (1). The cutting out operation from the resin was also performed in the same manner as (1). In this way, acOLLA-IKVAV was obtained.

  The obtained product was monitored by HPLC at 220 nm (absorption wavelength of peptide bond) and analyzed to confirm the introduction of a hydrophobic group.

(4) Production of electrospun nonwoven fabric A nonwoven fabric was produced by electrospinning. This fabrication method is schematically shown in FIG.

  A polymer solution (20 w / v%) was prepared using acOLLA-IKVAV (1 wt% / poly L lactic acid) obtained in (3) as a solute and hexafluoroisopropyl alcohol (HFIP) as a solvent. The polymer solution was taken in a plastic syringe 1 and extruded (13 kV, 3 mL / h) while applying voltage by a high voltage supply system 2 and spun onto a target 3. A stainless steel plate was used as the target 3, and spinning was performed for 10 minutes while rotating (109 rpm) as indicated by an arrow in FIG. Then, it dried under reduced pressure at room temperature for 2 to 3 days. The thickness of the nonwoven fabric was measured with a micrometer.

(5) Cell seeding experiment In the cell seeding experiment, rat dorsal root ganglion (DRG) cells were isolated and used. The spine was removed from the rat, and DRG was collected from the inside of the spine. Thereafter, nerve fibers were removed with a scalpel and placed in 800 μL of PBS (Ice-cold), followed by the following enzyme treatment.

  Enzyme treatment includes 100 μL of 2.5% Trypsin (final 0.25%), 3 μL of 70 U / μL DNase (final 200 U / mL), 100 μL of 1% collagenase (final 0.1%) (total 1 mL) The enzyme solution was used for 30 minutes at 37 ° C. (tapping every 2 to 3 minutes).

  Next, 9 mL of DMEM + 10% FBS was added, pipetted, passed through a cell strainer (40 μm), centrifuged (800 rpm, 6 minutes), and the supernatant was removed. Then, Sato medium (+ Fluorodeoxyuridine) was added and pipetted to isolate DRG cells.

Isolated DRG cells were seeded at 4 × 10 4 cells / cm ² . The nonwoven fabric produced in (3) was used as a sample. The nonwoven fabric was sterilized by UV irradiation for 30 minutes each, then put in a 24-well plate one by one, and fixed with a sterilized metal ring from above. The cells were cultured at 37 ° C. in a 5% CO ₂ atmosphere, and fixed with a neutral formalin buffer after 4 days. Thereafter, Giemsa staining was performed and observation was performed with a microscope. Then, neurite outgrowth cells were counted and assigned by the number of adherent cells. As a result, the neurite extension cell rate was 65%, and it was found that the nerve guide tube modified with the acOLLA-IKVAV conjugate can promote neurite extension.

(Comparative Example 1)
Instead of the blend of acOLLA-IKVAV conjugate and poly L lactic acid in (4) of Example 1, a non-woven fabric was produced by electrospinning only with poly L lactic acid. Otherwise, cell seeding experiments were conducted in the same manner as in Example 1. As a result, the neurite outgrowth cell rate was 49%, and it was found that this nerve induction tube did not promote neurite outgrowth much.

(Comparative Example 2)
Instead of the blend of acOLLA-IKVAV conjugate and poly-L lactic acid in Example 1 (4), a nonwoven fabric was prepared by electrospinning with only oligopeptide IKVAV and poly-L lactic acid. Otherwise, the cell seeding experiment was conducted in the same manner as in Example 1. As a result, the neurite outgrowth cell rate was 49%, and this nerve induction tube was improved with respect to the extension of the process compared to the nerve induction tube made of poly-L-lactic acid. I was not able to admit.

It is the figure which showed roughly the preparation method of the nonwoven fabric by electrospinning.

Explanation of symbols

1 Plastic syringe 2 High voltage supply system 3 Target

Claims (7)

Compound peptide and oligo lactic acid linked by a peptide bond, Ri name by molding a resin composition comprising,
The oligo lactic acid, nerve guidance tubes you express the following general formula 1 (m is an integer of 1 to 100.).

  The nerve guide tube according to claim 1, wherein the peptide has at least an Ile-Lys-Val-Ala-Val sequence. The nerve guide tube according to claim 1 or 2 , wherein the resin composition contains a biodegradable polymer. The nerve guide tube according to any one of claims 1 to 3 , wherein nerve cells grow along the inner wall of the tube. Oligolactic acid modified with a peptide comprising an oligolactic acid unit represented by the following general formula 2 and a peptide represented by the following general formula 3 (n is an integer of 1 to 500, o is 1 to 10) And A and B each represent a single bond or a divalent organic group.)

The molded object formed by shape | molding the resin composition containing the oligolactic acid modified with the peptide of Claim 5 . Molding the resin composition comprising the compound represented by the general formula 3 and the compound obtained by binding the oligolactic acid having the oligolactic acid unit represented by the general formula 2 by a peptide bond. The nerve guide tube according to any one of claims 1, 3, and 4 , wherein:

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