JP2009034374A - Nerve regeneration-inducing tube - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nerve regeneration-inducing tube with the use of collagen, which is excellent in nerve cell adhesiveness, cell growth properties and differentiation-inducing properties, as an anchorage for nerve regeneration. <P>SOLUTION: The nerve regeneration-inducing tube with the use of collagen as an anchorage for nerve regeneration, characterized in that the collagen employed has been purified so that the sodium chloride concentration thereof attains 2.0% by weight or less, preferably 0.1 to 1.5% by weight on the dry basis. The collagen is purified by isoelectric point precipitation at a pH value of 8 or more but less than 9. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a nerve regeneration inducing tube for reconnecting peripheral nerves cut or excised in an accident or surgery by utilizing the elongation of nerve cells. More specifically, the present invention uses collagen as a scaffold for nerve regeneration, which is used to fix the extending direction of a cut or excised nerve tissue and to join the cut sites without being disturbed by surrounding tissues. The present invention relates to a nerve regeneration induction tube.

  In many cases, peripheral nerve damage due to accidents cannot be repaired. In addition, there are many clinical cases in which peripheral nerves have to be excised with general surgery. For peripheral nerve damage, autologous nerve transplantation was the only measure other than direct anastomosis. However, the results were never satisfactory, the recovery of perception and motor ability was poor, and there were also sequelae due to erroneous control. In addition, there are many patients who suffer from not only aftereffects such as pain and sensory deficits, but also abnormal perception of the affected area, particularly pain.

  Attempts to regenerate nerves by connecting gaps in peripheral nerves using joint tubes made of artificial materials have been actively conducted since the early 1980s. However, research into bonded channels with non-absorbable synthetic artificial materials has failed. In order to solve this problem, it is necessary to prevent the invasion of connective tissue from the outside during regeneration of the nerve bundle, the exchange of substances inside and outside the channel or the formation of capillaries in the channel wall, Consideration must be given to the fact that a substance that can serve as a scaffold suitable for Schwann cell growth is necessary, and that the material used is decomposed and absorbed after regeneration. Considering these conditions, research on artificial nerve junction tubes using biodegradable and absorbable materials has been conducted since then.

  Regarding the regeneration of peripheral nerves, attempts have been made to extend the reproducible stump distance using silicone tubes since the introduction of the silicone tube model in 1982. However, the wall of the silicone tube cannot pass through nutrients, so there are problems such as insufficient supply of nutrients to nerve axons. Satisfactory nerve regeneration has not been obtained using Furthermore, even if the nerve can be regenerated, there is a problem that the silicone tube, which is a foreign substance, must be removed by re-operation or the like.

  On the other hand, regeneration of peripheral nerves using a tube made of a biodegradable polymer instead of a silicone tube has been attempted. If a nerve regeneration tube made of a biodegradable polymer is used, the nerve regeneration tube is gradually decomposed and absorbed in vivo by hydrolysis or the action of an enzyme after the nerve is regenerated. There is no need to take it out.

  As a nerve regeneration tube composed of such a biodegradable polymer, for example, Patent Document 1 discloses a nerve regeneration assisting material composed of a bundle of collagen fibers coated with laminin and fibronectin. Patent Document 2 includes a tube of a biodegradable absorbent material and a collagen body having a cavity penetrating the tube along the tube substantially parallel to the axis of the biodegradable absorbent material. The gap contains collagen, laminin, and the like. An artificial neural tube filled with a matrix gel containing is disclosed. Patent Document 3 discloses a tube of biodegradable absorbable material and an artificial neural tube in which a collagen fiber bundle covered with laminin is inserted into the lumen of the tube substantially parallel to the axis of the tube. Patent Document 4 discloses a nerve reconstruction base material having a structure in which fibers made of a bioabsorbable material are bundled. Patent Document 5 discloses a support such as a sponge, tube, or coil made of collagen. Patent Document 6 discloses a support comprising a sponge-like fine matrix made of a biodegradable material or a bioabsorbable material, and a linear biological tissue guide route or organ guide route. Further, Patent Document 7 discloses a nerve regeneration tube including a sponge made of a biodegradable polymer material and a reinforcing material made of a biodegradable polymer having a longer decomposition and absorption period than that of the sponge, the inner surface of which is made of a sponge. Yes.

All of these nerve regeneration tubes use collagen as a scaffold for nerve regeneration, but collagen adhesion, cell proliferation and differentiation-inducing ability of collagen are not sufficient.
JP-A-5-237139 WO98 / 22155 WO99 / 63908 JP 2000-325463 A JP 2001-70436 A JP 2002-320630 A Japanese Patent Laid-Open No. 2003-19196

  The present invention was devised in view of the current state of the prior art, and its purpose is to induce nerve regeneration using collagen excellent in nerve cell adhesion, cell proliferation and differentiation-inducing ability as a scaffold for nerve regeneration. To provide a tube.

  As a result of studying a method for producing collagen used as a scaffold for nerve regeneration in order to achieve such an object, the present inventor inevitably mixed during washing and salting out when producing collagen from raw materials such as pig skin. It has been found that sodium chloride has an adverse effect on nerve cell regeneration, and that the use of collagen with a reduced concentration of sodium chloride improves nerve cell proliferation and neurite outgrowth. The invention has been completed.

  That is, the present invention provides a nerve regeneration-inducing tube using collagen as a scaffold for nerve regeneration, so that the sodium chloride content is 2.0% by weight or less, preferably 0.1 to 1.5% by weight in a dry state. A nerve regeneration-inducing tube characterized by using purified collagen.

  In a preferred embodiment of the nerve regeneration-inducing tube of the present invention, collagen is purified by isoelectric point precipitation at a pH of 8 or more and less than 9, and the nerve regeneration-inducing tube coats a tubular body made of a biodegradable polymer with collagen, Furthermore, the inside of the tubular body is formed by filling collagen, the biodegradable polymer is selected from the group consisting of polyglycolic acid, polylactic acid, and lactic acid-caprolactone copolymer, and the tubular body has an inner diameter of 0.1 to 20 mm and an outer diameter. The diameter is 0.15 to 25 mm, and the length is 1.0 to 150 mm.

  The present invention uses a purified collagen in which the concentration of sodium chloride, which is inevitably mixed in the collagen production process, is reduced to 2% by weight or less, so that the adhesiveness, cell proliferation and differentiation inducing ability of nerve cells are achieved. It is possible to provide an excellent nerve regeneration induction tube.

  The nerve regeneration-inducing tube of the present invention is characterized by using collagen purified as a scaffold for nerve regeneration so that the sodium chloride-containing concentration mixed in the collagen production process is 2% by weight or less in a dry state.

  Collagen plays a role as a substrate for various cells, and therefore, when applied to a living body as a medical material, it has a good affinity with tissue and has been conventionally used as a scaffold for the growth of nerve cells.

  Conventional collagen used as a scaffold for nerve regeneration is generally made from pork skin collected and frozen at a meat inspection station, neutral protease is added to this, heated, and washed repeatedly with sodium chloride solution. After dehydration, wash with isopropanol and acetone, dry under reduced pressure to make a degreased chip, add this degreased chip into acetic acid solution, adjust pH with hydrochloric acid, add and decompose pepsin, sodium hydroxide solution Adjust to high pH (virus inactivation step 1), adjust to low pH with hydrochloric acid (virus inactivation step 2), adjust to pH 2-3 with sodium hydroxide and filter, add sodium chloride solution Salt out, concentrate by centrifugation, add and dissolve this concentrate in purified water, add sodium chloride solution again to salt out, concentrate by centrifugation, and freeze-dry. Thus it is produced.

  Thus, since conventionally used collagen includes washing with a sodium chloride solution and salting out with a sodium chloride solution in the production process, the sodium chloride concentration of the collagen used is 4% by weight, including a commercially available product. That was all. The present inventor considered that the concentration of sodium chloride in collagen affects the survival and growth of nerve cells, and if this concentration is too high, the cell membrane is destroyed by osmotic pressure. Therefore, when collagen purified so as to reduce the sodium chloride content concentration was used in the nerve regeneration induction tube, this nerve regeneration induction tube was superior to those using conventional collagen in cell adhesion, cell proliferation and differentiation. It has been found that it exhibits inductive ability. Based on this finding, the present invention has been purified to reduce the concentration of sodium chloride in a dry state as a scaffold for nerve regeneration-inducing tubes to 2.0% by weight or less, preferably 0.1 to 1.5% by weight. Use collagen. This sodium chloride concentration is measured by atomic absorption photometry (ashing). In order to prevent cell membrane destruction by lowering the osmotic pressure, the sodium chloride concentration should be low, but it is considered that the lower limit is about 0.1% by weight in terms of technical aspects and collagen stability. In addition, the measurement of sodium chloride concentration by atomic absorption spectrophotometry takes 1 to 4 g of a sample in a quartz beaker, gradually raises the temperature on an electric heater and carbonizes it, and finally takes 6 to 8 hours in a muffle furnace. Ashing (500 ° C.), the residue is redissolved with a 10% by weight aqueous hydrochloric acid solution, diluted to a final concentration of 1% by weight, and measured by flame atomic absorption spectrometry using acetylene-air. The measurement wavelength at this time is 589.6 nm.

  The collagen used in the nerve regeneration-inducing tube of the present invention can be produced by any conventionally known method. For example, the conventional collagen commercially available for medical use described above is used as a starting material, and this collagen is cooled at 2 to 10 ° C. Is dissolved in distilled water for injection, adjusted to pH 8 or more and less than 9 with sodium hydroxide solution, centrifuged, centrifuged, the supernatant discarded, and the precipitate freeze-dried. Can. The reason why the proliferation, adhesion, and differentiation inducing ability of cells are improved by using collagen having an isoelectric point of pH 8 or more and less than 9 as a scaffold for nerve regeneration has not been elucidated in detail. It is possible that the fraction that precipitates above contains a factor having low affinity with cells, and conversely, collagen that precipitates at pH 8 or more and less than 9 has particularly high affinity with cells. Conceivable. Alternatively, unpurified collagen is composed of type I collagen and type III collagen in a ratio of approximately 7: 3, and an effect due to the change in the composition ratio of type I and type III is considered.

  The nerve regeneration-inducing tube of the present invention can be produced according to a conventionally known method. For example, it is formed by coating a tubular body made of a biodegradable polymer with collagen and filling the inside of the tubular body with collagen. can do. The size of the tubular body is generally 0.1 to 20 mm in inner diameter, 0.15 to 25 mm in outer diameter, and 1.0 to 150 mm in length, although it depends on the part of the nerve to be regenerated and the required strength. Actually, it is preferable to prepare in advance a nerve regeneration guide tube made of a large number of types of tubular bodies in view of time constraints and production costs.

  Examples of the biodegradable polymer constituting the tubular body include polyglycolic acid, polylactic acid, lactic acid-caprolactone polymer, glycolic acid-caprolactone copolymer, polydioxanone, and glycolic acid-trimethylene carboxylic acid. From the viewpoint of easy availability and handling, it is preferable to use polyglycolic acid, polylactic acid, lactic acid-caprolactone copolymer, particularly polyglycolic acid. A biodegradable polymer may be used independently and may be used in mixture of 2 or more types.

  As the tubular body, a product obtained by molding the biodegradable polymer into a porous tubular body may be used, or a tubular body obtained by bundling a plurality of ultrafine fibers of the biodegradable polymer. It may be used. What is necessary is just to adjust suitably the pore diameter and porosity of a porous body or a stitch (mesh) according to the intended use and intensity | strength.

  An experiment demonstrating the superiority of the effect of the collagen of the present invention is shown below.

Experiment 1: Collagen gel culture experiment Purpose of this experiment Normally, two-dimensional culture on the bottom of a well plate is fundamental in cell culture experiments. However, when three-dimensional culture is performed, it is said that the behavior of cells during two-dimensional culture is greatly different, and if the ability to regenerate nerves is evaluated, three-dimensional culture is considered to be a system that is closer to reality. . Therefore, the purpose of this experiment was to perform three-dimensional culture with a collagen gel and check whether the behavior of cultured cells differs depending on the type of collagen.

2. Collagen used in this experiment (1) Collagen of Comparative Example “NMP Collagen PS” manufactured by Nippon Ham Co., Ltd. was used as collagen of Comparative Example. The collagen of this comparative example is produced by performing a degreasing process and a purification process using pig skin as a starting material, and the degreasing process includes a repeated washing step with a sodium chloride solution. Includes a salting-out step with sodium chloride. The collagen of this comparative example contained 4.0% by weight of sodium chloride in a dry state as measured by atomic absorption spectrophotometry (ashing).
(2) Collagen of the Invention Example A part of the collagen of the above Comparative Example was used as a starting material, which was purified by isoelectric precipitation at pH 8 or more and less than 9, to prepare the collagen of the invention example. This collagen of the present invention contained 1.0% by weight of sodium chloride in a dry state as measured by atomic absorption spectrophotometry (ashing).

3. Preparation of Collagen Gel Medium The two types of collagen prepared above were each dissolved in hydrochloric acid according to a conventional method to prepare a 0.5 wt% collagen-hydrochloric acid solution. Among these, 300 μl of the collagen solution of the present invention was added to 8 wells of a 24-well assay plate (manufactured by IWAKI), and 300 μl of the collagen solution of the comparative example was added to another 8 wells of the same plate. Thereafter, the plate was allowed to stand at 37 ° C. for 30 minutes in an incubator.

4). Collagen gel culture of PC12 cells (1) PC12 cells (cells derived from rat adrenal pheochromocytoma manufactured by Dainippon Pharmaceutical Laboratories) were previously cultured in DMEM medium to passage number 6, and the cells were collected by centrifugation, The cell number is adjusted to 1 × 10 ⁶ cells and suspended in 15 ml of DMEM medium, and 15 μl of NGF (cell growth factor, manufactured by R & D systems Inc., 50 μg / ml solution in phosphate buffered saline) is added. A culture solution was prepared.
The DMEM medium is RPMI 1640 liquid medium (Dainippon Pharmaceutical Laboratory Products, glutamic acid-free and sodium bicarbonate included), 500 ml of fetal calf serum (Dainippon Pharmaceutical Laboratories), and horse serum (Dainippon Pharmaceutical Laboratories). ) 5 ml of 50 ml, 200 mM glutamine solution (manufactured by Dainippon Pharmaceutical Laboratory Products, 29.23 mg / ml) is added and mixed.
(2) 300 μl of the prepared culture solution was dropped into wells of a collagen gel medium prepared in advance.
(3) The well plate was cultured in an incubator (37 ° C., CO ₂ concentration 5.0%) for 4 days.

5). Observation of cell appearance After 4 days of culture, the appearance of cells in the collagen gel was observed with a microscope, and representative examples were photographed. The results are shown in FIGS.

6). Measurement of the number of viable cells (1) To measure the number of viable cells after 4 days of culture, add 50 μl of 1 wt% collagenase solution to each well and gently stir the whole well with a collagen gel at 37 ° C. for 30 minutes. Dissolved.
(2) After dissolution of the collagen gel, 50 μl of the MTT assay solution was added to each well and allowed to stand for 30 minutes in an incubator.
(3) After standing for 30 minutes, the absorbance at 450 nm was measured, and for each collagen gel, the average value and the standard deviation were determined from the absorbance values in 8 wells, and the results are shown as a graph in FIG. In the graph of FIG. 3, the absorbance of the collagen of the example of the present invention is expressed as a relative value with the average absorbance of the collagen gel of the comparative example as 100. Absorbance is directly proportional to the number of viable cells.

7). Consideration of this experiment (1) Observation of the state of cells As is clear from the comparison of FIG. 1 and FIG. 2, in the culture using the collagen of the present invention example (FIG. 1), the culture using the collagen of the comparative example (FIG. 2). ) More proliferating cells and remarkable neurite outgrowth.
(2) Measurement of the number of viable cells As is clear from FIG. 3, the absorbance of the culture using the collagen of the example of the present invention is 39% larger on average than the absorbance of the culture using the collagen of the comparative example. There was also a significant difference (p <0.01). Therefore, it can be seen from the results of FIG. 3 that the collagen of the present invention example has a cell proliferation ability significantly superior to the collagen of the comparative example.
(3) From the above results, it can be said that the collagen of the example of the present invention is significantly superior in cell proliferation ability and differentiation inducing ability than the conventional collagen of the comparative example.

Experiment 2: Collagen coat adhesion experiment Purpose of this experiment To compare the cell adhesion of the collagen of the present invention and conventional collagen, after culturing with both collagens, suck up floating cells with an aspirator, measure only the cells that adhere to the collagen, and compare the number Therefore, it was aimed to confirm whether there was a significant difference in cell adhesion depending on the type of collagen.

2. Preparation of collagen coat (1) The collagen of the example of the present invention used in Experiment 1 and the collagen of the comparative example were diluted with hydrochloric acid so as to be 0.05% by weight, and each of 8 wells of a 24-well microplate (manufactured by IWAKI) 300 μl of these collagen solutions were added to the wells and allowed to stand in the refrigerator for 1 hour.
(2) After standing for 1 hour, the collagen solution in each well was sucked with an aspirator, and the collagen coat adhering to the well was naturally dried in a clean bench for 1 hour.

3. Collagen-coated culture of PC12 cells (1) PC12 cells (cells derived from rat adrenal pheochromocytoma manufactured by Dainippon Pharmaceutical Laboratories) were previously cultured in DMEM medium until passage number 6, and the cells were collected by centrifugation, The number of cells is adjusted to 5 × 10 ⁶ cells in 25 ml of DMEM medium and suspended, and 25 μl of NGF (cell growth factor, manufactured by R & D systems Inc., 50 μg / ml solution in phosphate buffered saline) is added. A culture solution was prepared.
(2) 300 μl of the prepared culture solution was added to each well of a collagen-coated well plate prepared in advance.
(3) The well plate was cultured in an incubator (37 ° C., CO ₂ concentration 5.0%) for 5 days.

4). Measurement of the number of adherent cells (1) In order to remove floating cells and cells that did not adhere properly, all the medium was aspirated while tilting the well plate at 85 degrees. At this time, care was taken not to suck adherent cells.
(2) Thereafter, 300 μl of DMEM medium and 30 μl of MTT assay solution were added to each well and allowed to stand for 30 minutes in an incubator.
(3) After standing for 30 minutes, the absorbance at 450 nm was measured, and the average value and standard deviation were determined from the absorbance values in 8 wells for each collagen coat, and are shown as a graph in FIG. In the graph of FIG. 4, the absorbance of the collagen coat of the present invention example is expressed as a relative value with the average absorbance of the collagen coat of the comparative example being 100.

5). Consideration of this experiment As is clear from FIG. 4, the absorbance representing the number of adherent cells of the collagen coat of the present invention example is 49% larger on average than the absorbance of the collagen coat of the comparative example, and this difference is also statistically significant. Difference (0.01 <p <0.05). The cell adhesion of the scaffold in regenerative medicine is a very important factor, and the results of FIG. 4 show that the collagen of the present invention is more suitable for use as a scaffold for nerve regeneration than conventional collagen.

Experiment 3: Collagen coat culture experiment with varying sodium chloride concentration The purpose of this experiment The purpose of this study was to examine how much the difference in sodium chloride content in collagen affects cell survival and growth.

2. Preparation of collagen coat (1) Collagen of the present invention used in Experiment 1, sodium chloride added thereto to adjust the sodium chloride concentration to 5% by weight and 10% by weight, Collagen of Comparative Example used in Experiment 1 Then, sodium chloride was added thereto to adjust the sodium chloride concentration to 5 wt% and 10 wt% (see collagen coats 1 to 6 in FIG. 5). Adjust each collagen to a 0.01 wt% hydrochloric acid solution, use two 24 well assay plates (manufactured by IWAKI), drop 300 μl of each collagen into 4 wells, and leave it in the refrigerator for 1 hour. did.
(2) After standing for 1 hour, the collagen solution in each well was sucked with an aspirator, and the collagen coat adhering to the well was naturally dried in a clean bench for 1 hour.

3. Collagen-coated culture of PC12 cells (1) PC12 cells (cells derived from rat adrenal pheochromocytoma manufactured by Dainippon Pharmaceutical Laboratories) were previously cultured in DMEM medium until passage number 6, and the cells were collected by centrifugation, The cell number is adjusted to 1 × 10 ⁶ cells and suspended in 15 ml of DMEM medium, and 15 μl of NGF (cell growth factor, manufactured by R & D systems Inc., 50 μg / ml solution in phosphate buffered saline) is added. A culture solution was prepared.
(2) 300 μl of the prepared culture solution was dropped into each well of a collagen-coated well plate prepared in advance.
(3) The well plate was cultured in an incubator (37 ° C., CO ₂ concentration 5.0%) for 5 days.

4). Measurement of the number of viable cells (1) 30 μl of the MTT assay solution was added to each well and allowed to stand in an incubator for 30 minutes.
(2) After standing for 30 minutes, the absorbance at 450 nm was measured, and the average value and standard deviation were determined from the absorbance values in 8 wells for each collagen coat, and are shown as graphs in FIGS. In the graphs of FIGS. 6 and 7, the absorbance of the collagen coat of the example of the present invention is expressed as a relative value with the average absorbance of the collagen coat of the comparative example as 100.

5). Discussion of this experiment As apparent from FIGS. 6 and 7, the absorbance tends to increase as the sodium chloride concentration of the collagen decreases, and the collagen of the present invention (collagen coat 1 (sodium chloride concentration of 1% by weight)) is used. The absorbance of the cultured medium was 27% larger on average than the absorbance of the culture using the collagen of the comparative example (collagen coat 6 (sodium chloride concentration 10% by weight)), and this difference was statistically significant. (P <0.01). From the results of FIGS. 6 and 7, it can be seen that the collagen of the present invention having a low sodium chloride concentration has a significantly superior cell growth property than the collagen of the comparative example.

  The nerve regeneration-inducing tube of the present invention is extremely useful because it is excellent in adhesiveness, cell proliferation, and differentiation-inducing ability of nerve cells, and thus has wide application in nerve regeneration medicine.

It is a microscope picture which shows the mode of the cell in the collagen gel of the example of this invention. It is a microscope picture which shows the mode of the cell in the collagen gel of a comparative example. 4 is a graph of absorbance (relative value) measured in Experiment 1. 5 is a graph of absorbance (relative value) measured in Experiment 2. Details of collagen coats 1 to 6 used in Experiment 3 are shown. 5 is a graph of absorbance (relative value) measured in Experiment 3. 5 is a graph of absorbance (relative value) measured in Experiment 3.

Claims (6)

  A nerve regeneration induction tube using collagen as a scaffold for nerve regeneration, wherein the collagen is purified so that the sodium chloride content is 2.0% by weight or less in a dry state.   2. The nerve regeneration-inducing tube according to claim 1, wherein collagen purified to have a sodium chloride content of 0.1 to 1.5% by weight in a dry state is used.   The nerve regeneration-inducing tube according to claim 1 or 2, wherein the collagen is purified by isoelectric point precipitation at a pH of 8 or more and less than 9.   The nerve regeneration-inducing tube according to any one of claims 1 to 3, which is formed by coating a tubular body made of a biodegradable polymer with collagen and filling the inside of the tubular body with collagen.   The nerve regeneration-inducing tube according to claim 4, wherein the biodegradable polymer is at least one polymer selected from the group consisting of polyglycolic acid, polylactic acid, and lactic acid-caprolactone copolymer.   6. The nerve regeneration induction tube according to claim 4, wherein the tubular body has an inner diameter of 0.1 to 20 mm, an outer diameter of 0.15 to 25 mm, and a length of 1.0 to 150 mm.