Scaffold material for use in nerve regeneration and / or angiogenesis

Abstract

[Problem] To provide a novel scaffold material that can be suitably used for regenerating nerves such as damaged peripheral nerves. [Solution] This scaffold material is for use in nerve regeneration and comprises a hydrogel having a structure in which a protein is crosslinked with a polyether through chemical bonding.

Description

Scaffold material for use in nerve regeneration and / or angiogenesis 【0001】 The present invention relates to a scaffold material for regenerating nerves such as damaged peripheral nerves and / or for creating new blood vessels, a kit for preparing the scaffold material, and a method for nerve regeneration using the scaffold material. 【0002】 When peripheral nerves are damaged due to accidents or other incidents, treatment involves either direct suturing through surgery or nerve grafting. However, this is difficult with crushed tissue, and nerve grafting from other parts of the body inevitably results in nerve loss in the donor nerve area. Furthermore, even when suturing is possible, recovery is only seen in about half of cases, and in many cases, nerve damage remains as a sequela. 【0003】 Traditionally, regarding peripheral nerve regeneration, a technique was developed in the 1980s to extend the distance between regenerative stumps using silicone tubes, which had the advantage of not requiring autologous tissue transplantation. However, with this technique, satisfactory nerve regeneration was not achieved because nutrients could not permeate the walls of the silicone tubes. 【0004】 In response to this, efforts have also been made to regenerate nerves using tubes made of biodegradable polymers instead of silicone tubes. For example, nerve regeneration aids have been proposed that include a bundle of collagen fibers coated with laminin and fibronectin attached to a biodegradable tube (Patent Document 1), and a support made of a biodegradable sponge-like fine matrix and a biological tissue induction pathway (Patent Document 2). 【0005】 While these nerve regeneration tubes can function as scaffolds for nerve regeneration, satisfactory results cannot be achieved unless the appropriate size tube is selected based on the size of the target nerve. In other words, the effectiveness may be compromised depending on the operator's judgment. From a resource perspective, it is wasteful to prepare a variety of sizes in advance. Furthermore, because suturing is required to connect the nerve end to the tube, there are limitations to its application for nerves that are too small to be sutured. 【0006】On the other hand, biodegradable hydrogels have also been developed as biocompatible materials applicable to various uses. However, the use of hydrogels in the field of nerve regeneration has been limited to in vitro applications such as scaffold materials for producing iPS cell-derived nerve cells. This is because there was no material that could completely fill nerve defects, solidify in situ at the appropriate time, and stably serve as a scaffold for nerve-related cells until nerve regeneration. For example, hydrogels consisting of physical cross-linking, such as collagen gels and alginate gels, are difficult to solidify at the appropriate time and tend to leak out of the application site before nerve regeneration, making them unable to function as a spatiotemporalally stable scaffold. 【0007】 Existing nerve regeneration therapies, including those mentioned above, have several drawbacks: they are not suitable for fine nerves such as cranial nerves, they require surgery and cannot be used for patients with high surgical risks, and existing tubular scaffold materials can only be used for discontinuous injuries. Therefore, there is a real need for new nerve regeneration therapies that overcome these challenges. 【0008】 Japanese Patent Publication No. 5-237139 Japanese Patent Publication No. 2002-320630 【0009】 Therefore, the object of the present invention is to provide a novel scaffold material that can be suitably used for regenerating nerves such as damaged peripheral nerves and / or for creating new blood vessels. 【0010】 As a result of diligent research to solve the aforementioned problems, the present inventors have found that hydrogels obtained by crosslinking proteins such as gelatin with a polyether crosslinking agent can be formed in situ and have appropriate voids that allow for the activity of nerve cells / tissues, making them extremely effective as scaffold materials for nerve regeneration, thus completing the present invention. They have also found that when nerve regeneration is performed using the above hydrogel, angiogenesis occurs around the regenerating nerve. Furthermore, they have also found that the nerve regeneration capacity can be further improved by further containing laminin in the hydrogel. Furthermore, they have also found that when nerve regeneration is performed using the above hydrogel, angiogenesis is promoted around the regenerating nerve. 【0011】 That is, in one aspect, the present invention relates to a scaffold material suitable for the treatment of nerve regeneration. More specifically, <1> a scaffold material for use in nerve regeneration and / or angiogenesis, comprising a hydrogel having a structure in which a protein is crosslinked to a polyether by a chemical bond; <2> the scaffold material according to <1> above, wherein the polyether is a polymer having a polyalkylene glycol backbone; <3> the scaffold material according to <1> above, wherein the polyether contains a 2-branched, 3-branched, 4-branched or 8-branched polyethylene glycol; <4> the scaffold material according to <1> above, wherein the polyether has a total of two or more electrophilic functional groups at the side chain or terminal; <5> the scaffold material according to <4> above, wherein the electrophilic functional group is selected from the group consisting of an N-hydroxysuccinimidyl (NHS) group, a maleimide group, a phthalimidyl group, an imidazolyl group, an acryloyl group, a nitrophenyl group, -CO ２ PhNO ２ , and a sulfosuccinimidyl group; <6> the scaffold material according to <1> above, wherein the protein is gelatin, collagen, albumin, fibrinogen, elastin, fibronectin, or a derivative thereof; <7> the scaffold material according to <1> above, wherein the polyether is present in the hydrogel in a ratio of 1 to 20% by weight; <8> the scaffold material according to <1> above, wherein the hydrogel contains laminin; <9> the scaffold material according to <7> above, wherein the laminin is a natural or recombinant protein. <10> the scaffold material according to <7> above, wherein the laminin is linked to the hydrogel by a chemical bond; <11> the scaffold material according to <7> above, wherein the laminin is present in the hydrogel in a ratio of 0.001 to 1% by weight; and <12> the scaffold material according to <1> above, wherein the nerve is a peripheral nerve. 【0012】In another aspect, the present invention relates to a kit for preparing the above scaffold material. More specifically, <13> a kit for preparing a scaffold material for use in nerve regeneration and / or angiogenesis, comprising at least solution A containing a protein and solution B containing a polyether, wherein the scaffold material comprises a hydrogel having a structure in which the protein is crosslinked to the polyether by a chemical bond by mixing the solutions A and B; <14> the kit according to <13> above, wherein the polyether is a polymer having a polyalkylene glycol backbone; and the protein is gelatin, collagen, albumin, fibrinogen, elastin, fibronectin, or a derivative thereof; and <15> the kit according to <13> above, wherein at least one of the solutions A and B further contains laminin, or further contains solution C containing laminin. 【0013】 In yet another aspect, the present invention relates to a method for nerve regeneration and a method for promoting angiogenesis using the above scaffold material. More specifically, <16> a method for nerve regeneration, comprising applying the scaffold material according to any one of <1> to <12> above around the damaged nerve; and <17> a method for promoting angiogenesis, comprising applying the scaffold material according to any one of <1> to <12> above around the damaged tissue. 【0014】 According to the scaffold material of the present invention, by having a structure in which a protein such as gelatin is crosslinked with a polyether crosslinking agent, it has appropriate voids that enable the activity of nerve cells / tissues, and efficient nerve regeneration can be achieved by directly applying it to the affected area of nerve damage. Also, in nerve regeneration treatment using the scaffold material of the present invention, it has been demonstrated that excellent recovery of motor function and angiogenesis around the regenerated nerve can also be provided. 【0015】 In addition, the scaffold material of the present invention not only can form a hydrogel in situ at the affected area, but also has fluidity such that it does not flow out of the affected area for about several weeks, and then has excellent properties of disappearing by biodegradation without inhibiting tissue regeneration. 【0016】Figure 1 is an illustrative image showing the procedure for each treatment on mice. Figure 2 is a graph showing the change in SFI over time. Figure 3 is a graph showing the improvement rate of SFI from 4 weeks. Figure 4 is a graph showing CMAP data at 0.5 mA stimulation. Figure 5 is a graph showing CMAP data at 1 mA stimulation. Figure 6 is a graph showing the contralateral ratio of muscle weight on the treated side at 24 weeks. Figure 7 shows a microscopic image of the area around regenerating nerves after applying PEG + gelatin + laminin cross-linked gel (left) and an image showing the results of immunostaining using CD31 (right). Figure 8 is a graph plotting the observed proliferation of CD31-positive vascular endothelial cells. 【0017】 Embodiments of the present invention will be described below. The scope of the present invention is not limited to these descriptions, and other embodiments may be modified and implemented as appropriate, as long as they do not impair the spirit of the invention. 【0018】 1. Scaffolding material of the present invention The scaffolding material of the present invention is suitable as a scaffolding material in nerve regeneration and / or angiogenesis, and is characterized by containing a hydrogel having a structure in which proteins are crosslinked with polyethers by chemical bonds. 【0019】 In the present invention, a hydrogel is a gel containing water as a dispersion medium. The amount of water contained in the hydrogel is, for example, 80 to 99% by mass, and more preferably 90 to 98% by mass. The dispersion medium is not limited to water, and a water-soluble organic solvent may be used in combination. Examples of water-soluble organic solvents include alcohols such as ethanol. Furthermore, it is preferable to use various aqueous buffer solutions or physiological saline solutions that can adjust the pH as the dispersion medium, and it is more preferable that phosphate-buffered physiological saline is included. 【0020】 A gel is generally a dispersion of a polymer that has lost its fluidity and high viscosity, and is a state in which the storage modulus G' and loss modulus G'' satisfy the relationship G'≧G''. Typically, it is a material with a three-dimensional network structure. The storage modulus G' and loss modulus G'' can be obtained by dynamic viscoelasticity testing using a rheometer. An example of measurement conditions is a temperature of 25°C, a shear amplitude of 1%, and a vibration frequency of 1.0 Hz. 【0021】 As described above, in the present invention, the hydrogel has a structure in which proteins are crosslinked with polyethers. Here, crosslinking refers to crosslinking by chemical bonds, typically covalent bonds. 【0022】 Furthermore, the hydrogel in this invention, having such a cross-linked structure, possesses sufficient fluidity to prevent leakage from the affected area when applied to a nerve injury site, and also exhibits biodegradability, where the cross-linked proteins are broken down by enzymes in the body, causing the gel to disappear after a certain period. This provides excellent retention at the affected area while simultaneously offering the excellent property of not inhibiting tissue regeneration. Preferably, the scaffold material of this invention can biodegrade several weeks to several months after being applied to the affected area. 【0023】 The proteins used in the present invention to constitute the above hydrogel include gelatin, albumin, collagen, fibrinogen, elastin, fibronectin, or derivatives thereof. Preferably, gelatin or albumin is used, and particularly preferably, gelatin. These proteins have multiple amino groups (-NH) in their molecules. ２ ) has such that the amino group reacts with the electrophilic functional group in the polyether described later to form a crosslink by chemical bonding. 【0024】 Gelatin is a widely used material in tissue engineering and cell therapy due to its availability, biocompatibility, and good cell adhesion. However, gelatin itself is highly soluble in water at 37°C and does not function as a support to retain material at the desired site. Therefore, in this invention, gelatin crosslinked with polyether is used. 【0025】 The amount of protein contained in the hydrogel is, for example, 0.5 to 6% by mass, preferably 0.5 to 4% by mass, and more preferably 1 to 3% by mass. 【0026】Next, the polyether used in the present invention to constitute the hydrogel is preferably a polymer having a structure in which alkylene glycols such as ethylene glycol and propylene glycol are polymerized. For example, this could be linear polyethylene glycol, linear polypropylene glycol, linear polybutylene glycol, or a polyether in which a structure in which alkylene glycol is polymerized to some or all of the hydroxyl groups of a polyhydric alcohol (e.g., glycerin, diglycerin, pentaerythritol, sorbitol, etc.) is bonded (e.g., polyethylene glycol chain, polypropylene glycol chain, polybutylene glycol chain, etc.). More preferably, the polyether used for crosslinking gelatin is a polyether having multiple polyalkylene glycol chains such as polyethylene glycol chain, polypropylene glycol chain, and polybutylene glycol chain (a so-called branched polyalkylene glycol). 【0027】 In this specification, the term "polyalkylene glycol" refers to both linear and branched polyalkylene glycols. Similarly, the term "polyethylene glycol" refers to both linear and branched polyethylene glycols. This also applies to "polypropylene glycol," etc. 【0028】 In this specification, the polyalkylene glycol skeleton refers to a structure in which the -RO- structural unit (where R is an alkylene group) is repeated, and this is also referred to as a "polyalkylene glycol chain." Furthermore, the polyethylene glycol skeleton refers to a structure consisting of -CH ２ CH ２ This refers to a structure in which O- structural units are repeated, and is also called a "polyethylene glycol chain." 【0029】The polyether is preferably a polymer having a polyethylene glycol (PEG) backbone. Therefore, in the present invention, the hydrogel is preferably composed of a protein crosslinked with a polymer having a polyethylene glycol backbone. The polyethylene glycol (PEG) is preferably a polymer having multiple polyethylene glycol backbones in its molecule. For example, a polymer having two polyethylene glycol backbones is called a two-branched PEG, a polymer having three polyethylene glycol backbones is called a three-branched PEG, a polymer having four polyethylene glycol backbones is called a four-branched PEG, a polymer having eight polyethylene glycol backbones is called an eight-branched PEG, and so on. A polymer having multiple polyethylene glycol backbones in its molecule is also called a "multi-armed PEG". 【0030】 Gels consisting of a tetrabranched polyethylene glycol skeleton are generally known as Tetra-PEG gels. A network structure is constructed by an AB-type cross-end coupling reaction between two tetrabranched polymers, each having electrophilic functional groups such as active ester structures and nucleophilic functional groups such as amino groups at their ends (Matsunaga et al., Macromolecules, Vol. 42, No. 4, pp. 1344-1351, 2009). Furthermore, Tetra-PEG gels can be easily prepared in situ by simply mixing two polymer solutions, and the gelation time can be controlled by adjusting the pH and ionic strength during gel preparation. Moreover, because this gel is mainly composed of PEG, it also exhibits excellent biocompatibility. 【0031】 The polyether used in the present invention preferably contains bibranched, tribranched, tetrabranched, or octabranched polyethylene glycol. 【0032】In the present invention, the polyether used for forming the hydrogel preferably has an electrophilic functional group in order to easily form a chemical bond with the amino acid residue of the protein. In particular, the polyether can have a total of two or more electrophilic functional groups in the side chain or at the terminal. The polyether can have a hydroxyl group derived from the raw material, and by utilizing this hydroxyl group, an electrophilic functional group can be easily introduced. The electrophilic functional group is preferably introduced at the terminal of a structure in which an alkylene glycol such as a polyethylene glycol skeleton is polymerized via a linker site, if necessary. 【0033】 Examples of such electrophilic functional groups include a maleimide group, an N-hydroxysuccinimidyl (NHS) group, a succinimidyl carbonate group, a sulfo succinimidyl group, a phthalimidyl group, an imidazolyl group, an acryloyl group, a nitrophenyl group, -CO ２ PhNO ２ (Ph represents an o-, m-, or p-phenylene group), etc., and those skilled in the art can appropriately use known electrophilic functional groups. Preferably, the electrophilic functional group is selected from the group consisting of an N-hydroxysuccinimidyl group, a maleimide group, and a sulfo succinimidyl group. When the polyether used for crosslinking gelatin has a plurality of electrophilic functional groups, the electrophilic functional groups may be the same or different from each other, but it is preferable that they are the same. 【0034】 When the protein is gelatin, the polyether preferably has an electrophilic functional group selected from the group consisting of an N-hydroxysuccinimidyl group, a maleimide group, and a sulfo succinimidyl group, and from the viewpoint of binding to the lysine residue of gelatin, it is particularly preferable to have an N-hydroxysuccinimidyl (NHS) group. 【0035】 The polyether used in the present invention preferably has a weight average molecular weight in the range of 2×10 ３ to 1×10 ５ More preferably, it is in the range of 5×10 ３ to 4×10 ４ Even more preferably, it is in the range of 1×10 ４ to 2×10 ４This is within the range. In this specification, the weight-average molecular weight of a polyether is the weight-average molecular weight on a polyethylene glycol basis, calculated by gel permeation chromatography (GPC). 【0036】 Preferred non-limiting specific examples of PEG having electrophilic functional groups at the terminals include, for example, the compound represented by the following formula (I), which has four polyethylene glycol skeletons, each polyethylene glycol skeleton having an N-hydroxysuccinimidyl (NHS) group at its terminal via a linker moiety. 【0037】 In the above formula (I), R ２１ ~R ２４ These are either the same or different (preferably the same), C １ -C ７ Alkylene group, C ２ -C ７ Alkenylene group, -NH-R ２５ -, -CO-R ２５ -, -R ２６ -O-R ２７ -, -R ２６ -NH-R ２７ -, -R ２６ -CO ２ -R ２７ -, -R ２６ -CO ２ -NH-R ２７ -, -R ２６ -CO-R ２７ -, or -R ２６ -CO-NH-R ２７ This indicates -. Here, R ２５ is C １ -C ７ R indicates an alkylene group. ２６ is C １ -C ３ It shows an alkylene group, R ２７ is C １ -C ５ R indicates an alkylene group. ２１ ~R ２４ This is a linker moiety that connects the N-hydroxysuccinimidyl (NHS) group to the polyethylene glycol skeleton. 【0038】 In the above formula (I), n ２１~n ２４ These may be the same or different. ２１ ~n ２４ The closer the values ​​are, the better, and it is especially preferable that they are the same. ２１ ~n ２４ Examples include integer values ​​from 11 to 569, preferably from 28 to 228, and more preferably from 56 to 114. 【0039】 In this specification, "C １ -C ７ An alkylene group refers to an alkylene group with 1 to 7 carbon atoms, which may have branching, and is a straight-chain C group. １ -C ７ C with an alkylene group or one or more branches ２ -C ７ This refers to an alkylene group (a group with 2 to 7 carbon atoms, including branching). １ -C ７ Examples of alkylene groups include -CH2-, -(CH2)2-, -(CH2)3-, -CH(CH3)-, -(CH2)3-, -(CH(CH3))2-, -(CH2)2-CH(CH3)-, -(CH2)3-CH(CH3)-, -(CH2)2-CH(C2H5)-, -(CH2)6-, -(CH ２ Examples include )2-C(C2H5)2- and -(CH2)3C(CH3)2CH2-. Note that "C １ -C ３ The term "alkylene group" refers to an alkylene group having 1 to 3 carbon atoms, which may have branching, and is defined as "C １ -C ５ The term "alkylene group" refers to an alkylene group having 1 to 5 carbon atoms, which may have branching. 【0040】 In this specification, "C ２ -C ７ An "alkenylene group" is a linear or branched alkenylene group having 2 to 7 carbon atoms and having one or more double bonds in the chain. For example, a divalent group has one or more double bonds formed by removing hydrogen atoms from each of the adjacent carbon atoms of the alkylene group. 【0041】In this specification, alkylene groups and alkenylene groups may have one or more substituents. Examples of substituents include, but are not limited to, alkoxy groups, halogen atoms (which may be fluorine, chlorine, bromine, or iodine atoms), amino groups, mono- or disubstituted amino groups, substituted silyl groups, acyl groups, or aryl groups. If alkylene groups and alkenylene groups have two or more substituents, the substituents may be the same or different. Furthermore, if the substituents include hydrocarbon groups (e.g., alkoxy groups, acyl groups, aryl groups, etc.), the hydrocarbon groups may also similarly have one or more substituents. 【0042】 Furthermore, in this specification, when a functional group is defined as "may have substituents," the type of substituent, the position of substitution, and the number of substituents are not particularly limited, and if there are two or more substituents, they may be the same or different. Examples of substituents include, but are not limited to, alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, and oxo groups. These substituents may have further substituents. 【0043】 The polyether is preferably present in the hydrogel in a ratio of 1 to 20% by weight, more preferably 2 to 10% by weight. This weight percentage is the weight ratio to the total amount of the hydrogel, including the polyether, protein, and solvent. 【0044】 In a preferred embodiment, the hydrogel in the scaffold material of the present invention may contain laminin. As shown in the examples described below, the inclusion of such laminin can provide the advantage of further improving nerve regeneration capacity. 【0045】The laminin in question may be natural, purified, or a genetically modified protein. Laminin is a heterotrimer molecule consisting of three subunit chains: an α chain, a β chain, and a γ chain. Five types of α chains (α1-α5), three types of β chains (β1-β3), and three types of γ chains (γ1-γ3) are known. More than 12 laminin isoforms exist due to their combinations. For example, laminin 221 and laminin 211 are known to bind to the integrin α7X2β1 protein, which is selectively expressed in muscle tissue. It has been reported that laminin 221 has a higher binding affinity to integrin α7X2β1 than laminin 211. Integrin α7X2β1 has been reported to be expressed in cardiomyocytes and skeletal muscle cells. As an example of a purified laminin product, we can mention "iMatrix-221," which is a highly purified integrin-binding site (E8 fragment) of the human laminin 221 protein. 【0046】 Laminin can preferably be present in the hydrogel in a ratio of 0.001 to 1% by weight, more preferably 0.001 to 0.01% by weight. Preferably, the laminin can be linked to the hydrogel by chemical bonds. 【0047】 2. In a kit-specific embodiment of the present invention, the present invention also relates to a kit suitable for preparing the above-mentioned scaffolding material. 【0048】 The kit of the present invention contains raw materials for forming a hydrogel having a structure in which a protein is crosslinked with a polyether by chemical bonds, and specifically comprises at least a solution A containing a protein and a solution B containing a polyether. 【0049】 The protein concentration in solution A is preferably in the range of 5 to 60 g / L, more preferably in the range of 5 to 40 g / L, and even more preferably in the range of 10 to 30 g / L. 【0050】 The concentration of polyether in solution B is preferably in the range of 10 to 200 g / L, and more preferably in the range of 20 to 100 g / L. 【0051】The pH of solution A and solution B is preferably in the range of 6.5 to 11.5, more preferably in the range of 7.0 to 10.5, and even more preferably in the range of 7.0 to 8.5. 【0052】 Solutions A and B contain water as a solvent, but in some cases, they may also be mixed solvents containing alcohols such as ethanol or other organic solvents. Preferably, polymer solutions A and B are aqueous solutions with water as the sole solvent. The volumes of polymer solutions A and B can be appropriately adjusted depending on the area and structural complexity of the affected area to which they are applied, but typically they are in the range of 0.1 to 20 ml each, preferably 1 to 10 ml. 【0053】 The pH of solutions A and B can be adjusted using pH buffers known in the art. For example, the pH can be adjusted to the above range using McIlbain buffer (citric acid phosphate buffer; CPB) or 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer. 【0054】 Preferably, solution A and solution B are mixed so that the ratio of protein to polyether is in the range of 0.25 to 11.5 by weight (polyether / protein). 【0055】 The temperatures of solution A and solution B during mixing should be such that the protein and polyether are dissolved in each solution and both solutions A and B are fluid. The temperatures of solution A and solution B during mixing may be different, but it is preferable to mix them at the same temperature. 【0056】The preferred method for producing a hydrogel involves mixing solution A, which contains protein, with solution B, which contains polyether. By adjusting the concentrations of protein and polyether in the solutions, as well as the pH of the solutions, the time from mixing solution A and solution B until gelation occurs (i.e., the time until the storage modulus G' equals the loss modulus G'') can be appropriately adjusted. In this specification, this is referred to as the "gelation time," and gelation may also be referred to as solidification. The gelation time is preferably in the range of 10 to 300 seconds, and more preferably in the range of 30 to 100 seconds. 【0057】 Polymer solutions A and B may be applied independently to the area around the nerve, or a solution may be prepared by mixing polymer solutions A and B immediately beforehand, and then this mixed solution may be applied to the area around the nerve. This allows for in-situ hydrogel formation. 【0058】 The polymer solutions A and B can be mixed using, for example, a two-component mixing syringe as disclosed in International Publication WO2007 / 083522. The temperatures of the two liquids during mixing are not particularly limited, and should be such that the precursor units are dissolved and each liquid is fluid. For example, the temperatures of the two liquids may be different, but it is preferable that they be at the same temperature as this facilitates mixing. 【0059】 Furthermore, it is preferable that the scaffold material containing hydrogel formed around the nerve remains around the affected area for the period necessary for nerve repair and regeneration. For example, the scaffold material of the present invention preferably has a degradation rate of 10 to 90 days in vivo, and more preferably a degradation rate of 20 to 50 days. 【0060】 In a preferred embodiment, at least one of solutions A and B may further contain laminin. Alternatively, the kit of the present invention may further contain a solution C that separately contains laminin. 【0061】 Details of the protein, polyether, and laminin included in the kit of the present invention are as described above. 【0062】Furthermore, the kit of the present invention may further include a medical device for covering the area around the nerve covered by the scaffolding material. Such a medical device is suitable for stably fixing the nerve and may, for example, be a device that can form a hollow cylindrical shape. However, a device of an appropriate shape may be used depending on the location of the affected area and the postoperative condition. 【0063】 2. Methods for nerve regeneration and angiogenesis of the present invention In a further embodiment, the present invention also relates to a method for nerve regeneration, comprising applying the above-described scaffold material around a damaged nerve. In another aspect, the present invention also relates to a method for promoting angiogenesis, comprising applying the above-described scaffold material around damaged tissue. 【0064】 As described above, for example, the kit of the present invention can be used to form a hydrogel in situ around a damaged nerve and cover the target nerve area. In this specification, nerves include nerve tissue that makes up the central nervous system and the peripheral nervous system, but typically refers to nerve tissue of the peripheral nervous system. 【0065】 Angiogenesis refers to the phenomenon in which new blood vessels are formed from existing blood vessels in tissues and organs. Furthermore, "promotion of angiogenesis" encompasses an increase in the frequency of new blood vessel formation, an increase in the final number of new blood vessels, an increase in the final length of new blood vessels, an increase in the thickness of formed blood vessels, and an increase in blood flow in collateral circulation. Since vascular endothelial cells (including their precursor cells) are known to construct tubular structures and form a vascular network through which fluids, especially blood, pass, it can be said that angiogenesis is promoted when vascular endothelial cells proliferate around a particular tissue. In this invention, it was confirmed that by applying the above-mentioned scaffold material around a damaged nerve, nerve regeneration and proliferation of vascular endothelial cells can be achieved. In addition, the CD31 protein is known as a cell surface marker for vascular endothelial cells, and the presence of vascular endothelial cells in living tissue can be detected by detecting the CD31 protein using any method. 【0066】 The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. 【0067】 1. Preparation of raw materials: As the raw material polyether, a 4-branched PEG commercially available from SINOPEG was used. Specifically, these are four-branched PEGs having succinimidyl groups at the ends (Tetra-PEG-SC (tetrasuccinimidyl-polyethylene glycol) (molecular weight 20,000)), four-branched PEGs having sulfhydryl groups at the ends (Tetra-PEG-SH (tetrasulfhydryl-polyethylene glycol) (molecular weight 10,000)), four-branched PEGs having maleimide groups at the ends (Tetra-PEG-MAL (tetramaleimide-polyethylene glycol) (molecular weight 10,000)), four-branched PEGs having diols at the ends (Tetra-PEG-GDL (tetradiol-polyethylene glycol) (molecular weight 20,000)), and four-branched PEGs having phenylboronic acid at the ends (Tetra-PEG-FPBA (tetraphenylboronic acid-polyethylene glycol) (molecular weight 20,000)). Nippi gelatin (product name "High-Grade Gelatin (APAT)") was used as the raw material protein. This gelatin was processed for 30 A gelatin solution was prepared by adding D-PBS(-) (manufactured by Fujifilm Wako Pure Chemical Industries) to a concentration of g / L, and stored in a refrigerator until immediately before use. As laminin, the product name "iMatrix-221," which is a highly purified product of the integrin binding site (E8 fragment) of the human laminin 221 protein, was used as is. As a buffer for the raw material solution, McIlbain buffer (also known as citrate phosphate buffer, CPB) or HEPES buffer (manufactured by Dojin Chemical Laboratories) was used, adjusted to the respective pH and concentration. A Minisart(R) RC15-AC (Sartorius Stedim) was used as a 0.22 μm filter. 【0068】2. Preparation of Applicable Materials Example 1 Gelatin-PEG Crosslinked Gel 680 μL of gelatin solution was added to an empty 1.5 mL tube. 170 μL of D-PBS(-) was added and mixed with a vortex mixer. This was solution A (850 μL) with a gelatin concentration of 24 g / L. Solution A was filtered through a 0.22 μm filter. 400 μL of HEPES 200 mM (pH 8.2) was added to a tube containing 40 mg of Tetra-PEG-SC powder and stirred with a vortex mixer. This was solution B with a PEG concentration of 100 g / L. Solution B was filtered through a 0.22 μm filter. A new empty 1 mL tube was prepared, and 100 μL of filtered solution A was taken into it. 100 μL of filtered solution B was added to the same tube and quickly mixed with a vortex mixer. This was used to create a gelling solution (200 μL) with a final concentration of [gelatin 12 g / L, PEG 50 g / L]. The entire gelling solution was filled into a 1 mL syringe without a needle, and approximately 100 μL was dispensed onto the affected area. Gelation was visually confirmed, and the wound was closed. 【0069】 Example 2 Gelatin-PEG-Laminin Crosslinked Gel 680 μL of gelatin solution was added to an empty 1.5 mL tube. 170 μL of laminin was added and mixed with a vortex mixer. This was solution A (850 μL) with a gelatin concentration of 24 g / L and a laminin concentration of 0.1 g / L. Solution A was filtered through a 0.22 μm filter. 400 μL of HEPES 200 mM (pH 8.2) was added to a tube containing 40 mg of Tetra-PEG-SC powder and mixed with a vortex mixer. This was solution B with a PEG concentration of 100 g / L. Solution B was filtered through a 0.22 μm filter. A new empty 1.5 mL tube was prepared and 100 μL of filtered solution A was placed in it. 100 μL of filtered solution B was added to the same tube and quickly mixed with a vortex mixer. This was used to create a gelling solution (200 μL) with the final concentration [gelatin 12 g / L, laminin 0.05 g / L, PEG 50 g / L]. The entire gelling solution was filled into a 1 mL syringe without a needle, and approximately 100 μL was dispensed to the affected area. Gelation was visually confirmed, and the wound was closed. 【0070】Comparative Example 1: Sciatic nerve transection only. An appropriate amount of D-PBS(-) was filtered through a 0.22 μm filter. At least 150 μL of the filtered D-PBS(-) was filled into a 1 mL syringe without a needle, and approximately 100 μL was dispensed to the affected area. The wound was immediately closed. 【0071】 Comparative Example 2: 1035 μL of D-PBS(-) was added to an empty 1.5 mL tube containing laminin aqueous solution. 115 μL of laminin was then added and mixed using a vortex mixer. This resulted in 1150 μL of an injection solution containing 0.05 g / L of laminin. The injection solution was filtered through a 0.22 μm filter. At least 150 μL of the filtered injection solution was filled into a 1 mL syringe without a needle, and approximately 100 μL was dispensed into the affected area. The wound was immediately closed. 【0072】 Comparative Example 3 PEG Gel (containing laminin) To a tube containing approximately 10 mg of Tetra-PEG-SH powder, CPB 10 mM (pH 5.8) was added to a total concentration of 12.5 g / L and mixed with a vortex mixer (e.g., 800 μL per 10 mg). In an empty 1.5 mL tube, 360 μL of PEG-SH solution and 90 μL of laminin were mixed to prepare 450 μL of [PEG-SH 10 g / L, laminin 0.1 g / L] solution. To a tube containing approximately 10 mg of Tetra-PEG-MAL powder, CPB 10 mM (pH 5.8) was added to a total concentration of 10 g / L and mixed with a vortex mixer (e.g., 1,000 μL per 10 mg). The [PEG-SH 10 g / L, laminin 0.1 g / L] solution and the PEG-MAL solution were each filtered through a 0.22 μm filter. An empty 1.5 mL tube was prepared, and 100 μL of the [PEG-SH 10 g / L, laminin 0.1 g / L] solution was placed in it. 100 μL of the PEG-MAL solution was added to the same tube, and the mixture was quickly mixed using a vortex mixer. This resulted in 200 μL of a [PEG 10 g / L + laminin 0.05 g / L] gel solution. The gel solution was completely filled into a 1 mL syringe without a needle, and approximately 100 μL was dispensed to the affected area. After visual confirmation of gelation, the wound was closed. 【0073】Comparative Example 4 PEG Slime (containing laminin) D-PBS(-) was added to a tube containing approximately 20 mg of Tetra-PEG-GDL powder to a total concentration of 25 g / L and mixed with a vortex mixer (e.g., 800 μL per 20 mg). 520 μL of Tetra-PEG-GDL solution and 130 μL of laminin were mixed in an empty 1.5 mL tube to prepare 650 μL of [Tetra-PEG-GDL 20 g / L, laminin 0.1 g / L]. D-PBS(-) was added to a tube containing approximately 20 mg of Tetra-PEG-FPBA powder to a total concentration of 20 g / L and mixed with a vortex mixer (e.g., 1000 μL per 20 mg). The [PEG-GDL 20 g / L, laminin 0.1 g / L] solution and the PEG-FPBA solution were each filtered through a 0.22 μm filter. An empty 1.5 mL tube was prepared, and 200 μL of the [PEG-GDL 20 g / L, laminin 0.1 g / L] solution was placed in it. 200 μL of the PEG-FPBA solution was added to the same tube, and the mixture was quickly mixed using a vortex mixer. A slime-like substance was immediately formed upon mixing. This was used to make 400 μL of [PEG 20 g / L, laminin 0.05 g / L] slime. The plunger rod of a 1 mL syringe without a needle was removed, and only the solid component of varying concentration was filled into the syringe using tweezers. Approximately 100 μL was dispensed into the affected area without connecting a needle. The wound was then immediately closed. 【0074】 2. Application to the mouse model A mouse (C57BL / 6J strain, 8 weeks old, body weight 25-30 g, male) was used. A group that received no treatment was designated as the untreated group. Under inhalation anesthesia with isoflurane, the skin was incised and the right sciatic nerve was exposed. At this time, no pocket was made, and the sciatic nerve was exposed by cutting, and the exposed sciatic nerve was excised by 10 mm. A group that did not apply any special material to the excision site was designated as the sciatic nerve transection only group (Comparative Example 1). The groups to which materials were applied were gelatin-PEG crosslinked gel (Example 1), gelatin-PEG-laminin crosslinked gel (Example 2), laminin aqueous solution (Comparative Example 2), PEG gel (containing laminin) (Comparative Example 3), and PEG slime (containing laminin) (Comparative Example 4). After the procedure, subcutaneous suturing was not performed, and the entire thickness was sutured with 4-5 stitches. An example of these series of procedures is shown in Figure 1. 【0075】 The sciatic function index (SFI) was measured at each time point as an indicator of motor function recovery. In the group with only sciatic nerve transection, the value continued to decline, while in the gelatin-PEG cross-linked gel and gelatin-PEG-laminin cross-linked gel groups, the value declined towards week 4 but improved towards week 24 (Figures 2 and 3). At this time, the gelatin-PEG-laminin cross-linked gel showed the best improvement. 【0076】 2-1 SFI Measurement Protocol Red water-based ink was applied to both forelegs of the mice, and black water-based ink (non-toxic) was applied to the hind legs. The mice were then made to walk on white paper, and foot prints were collected. PL (distance from the heel to the third toe), TS (distance from the first to the fifth toe), and IT (distance from the second to the fourth toe) were measured for consecutive right lower limbs (affected side) and left lower limbs (unaffected side), and SFI was calculated using the following formula. SFI = -38.3 × (EPL - NPL) / NPL + 109.5 × (ETS - NTS) / NTS + 13.3 × (EIT - NIT) / NIT - 8.8 Here, "EPL", "ETS", and "EIT" are the PL, TS, and IT values ​​of the experimental group, respectively, and "NPL", "NTS", and "NIT" are the PL, TS, and IT values ​​of the normal group, respectively. 【0077】 As an indicator of nerve regeneration, the compound muscle action potential (CMAP) was evaluated. Low values ​​were observed in the sciatic nerve transection group and the laminin aqueous solution group, while changes in potential were observed in the gelatin-PEG cross-linked gel and the gelatin-PEG-laminin cross-linked gel when stimulated with 0.5 mA and 1 mA (Figures 4 and 5). At this time, the gelatin-PEG-laminin cross-linked gel showed the largest change in potential. This indicates that the regenerated tissue conducted electricity as nerves. 【0078】2-2 CMAP Measurement Protocol The skin was incised and the bilateral sciatic nerves were re-exposed. Stimulation electrodes were placed on the proximal side of the resected sciatic nerves and electrical stimulation was performed, while observation electrodes placed on the distal side were used to record CMAP. Stimulation and measurement were performed using ADINSTRUMENTS PowerLab 26T04. Stimulation consisted of three consecutive 1 Hz pulses as one set, with appropriate intervals between sets, for a total of three sets. The pulse width of each pulse was set to 50 μs. The stimulation intensity was set to 1.0 mA and 0.5 mA, and the maximum amplitude of the CMAP was calculated. The ratio (Cr) of affected side (Ci) / unaffected side (Cc) was calculated using the following formula and compared between each group: Cr = Ci / Cc × 100 (%) 【0079】 Compared to the groups treated with sciatic nerve transection alone and laminin aqueous solution, the degree of muscle atrophy was suppressed in the gelatin-PEG crosslinked gel and gelatin-PEG-laminin crosslinked gel (Figure 6). 【0080】 2-3 Muscle Atrophy Assessment Protocol The skin was incised to expose both gastrocnemius muscles. The gastrocnemius muscles were isolated by cutting the muscle attachments at their origin and insertion. After removing connective tissue and vascular tissue, the muscles were washed with D-PBS(-) to remove blood and other debris, wiped dry, and measured for wet weight. The ratio (Wr) of affected side (Wi) to unaffected side (Wc) was calculated using the following formula and compared between the groups: Wr = Wi / Wc × 100 (%). 【0081】 The results of nerve regeneration ability obtained in Examples 1-2 and Comparative Examples 1-4 are shown in the following comparison table. 【0082】 【0083】In Examples 1 and 2, nerve regeneration ability was observed based on SFI, CMAP, and muscle atrophy levels. On the other hand, in Comparative Examples 3 and 4, no signs of nerve regeneration were observed in the PEG gel and PEG slime, even when laminin was included. Furthermore, in the laminin aqueous solution of Comparative Example 2, although some improvement in SFI was observed, it is considered that nerve regeneration did not occur from the perspective of CMAP and muscle atrophy. These results indicate that simply including laminin in an in-situ hydrogel does not guarantee nerve regeneration for everyone; in other words, the combination of gelatin-PEG-laminin crosslinked gel is the best. 【0084】 3. Immunohistological evaluation of regenerated tissue (regenerated tissue findings, CD31 staining) In addition to evaluating the regenerated nerve after treatment, angiogenesis within the regenerated tissue was evaluated. The experimental procedure was the same as the histological evaluation in 2 above, with gelatin-PEG crosslinked gel (Example 1), gelatin-PEG-laminin crosslinked gel (Example 2), laminin aqueous solution (Comparative Example 2), and PBS solution (Comparative Example 1: sciatic nerve transection only) being applied to the sciatic nerve transection site. Immunostaining was performed on the sciatic nerve after application using CD31, a marker for vascular endothelial cells, to evaluate vascular infiltration within the regenerated tissue. Evaluations were performed at 12 and 24 weeks post-treatment, and significant angiogenesis was observed in the PEG + gelatin + laminin crosslinked gel group at both stages (both p < 0.01). 【0085】 Microscopic examination of the regenerated tissue revealed the formation of vascular-like structures around the regenerated nerves (Figure 7 left). Histologically, the PEG + gelatin + laminin cross-linked gel group showed proliferation of CD31-positive vascular endothelial cells around the regenerated nerves (Figure 7 right). Figure 8 shows a plot of the observed proliferation of CD31-positive vascular endothelial cells.

Claims

1. A scaffold material for use in nerve regeneration and / or angiogenesis, comprising a hydrogel having a structure in which proteins are crosslinked with polyethers by chemical bonds.

2. The scaffolding material according to claim 1, wherein the polyether is a polymer having a polyalkylene glycol backbone.

3. The scaffolding material according to claim 1, wherein the polyether comprises a bifurcated, trifurcated, quadrifurcated, or octufurcated polyethylene glycol.

4. The scaffolding material according to claim 1, wherein the polyether has a total of two or more electrophilic functional groups in its side chains or terminals.

5. The electrophilic functional groups include N-hydroxysuccinimidyl (NHS) group, maleimide group, phthalimidyl group, imidazoyl group, acryloyl group, nitrophenyl group, and -CO ２ PhNO ２ The scaffolding material according to claim 4, selected from the group consisting of and a sulfosuccinimidyl group.

6. The scaffolding material according to claim 1, wherein the protein is gelatin, collagen, albumin, fibrinogen, elastin, fibronectin, or a derivative thereof.

7. The scaffolding material according to claim 1, wherein the polyether is present in the hydrogel in a ratio of 1 to 20% by weight.

8. The scaffolding material according to claim 1, wherein the hydrogel contains laminin.

9. The scaffolding material according to claim 7, wherein the laminin is a natural or genetically modified protein.

10. The scaffolding material according to claim 7, wherein the laminin is linked to the hydrogel by chemical bonding.

11. The scaffolding material according to claim 7, wherein the laminin is present in the hydrogel in a ratio of 0.001 to 1% by weight.

12. The scaffolding material according to claim 1, wherein the nerve is a peripheral nerve.

13. A kit for preparing scaffold material for use in nerve regeneration and / or angiogenesis, comprising at least a solution A containing a protein and a solution B containing a polyether, wherein the scaffold material comprises a hydrogel having a structure in which the protein is crosslinked with the polyether by chemical bonds when the solutions A and B are mixed.

14. The kit according to claim 13, wherein the polyether is a polymer having a polyalkylene glycol backbone; and the protein is gelatin, collagen, albumin, fibrinogen, elastin, fibronectin, or a derivative thereof.

15. The kit according to claim 13, wherein at least one of the solutions A and B further comprises laminin, or further comprises a solution C containing laminin.

16. A method for nerve regeneration, comprising applying a scaffolding material according to any one of claims 1 to 12 around a damaged nerve.

17. A method for promoting angiogenesis, comprising applying a scaffolding material according to any one of claims 1 to 12 around damaged tissue.

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