JP2007204881A - Vitrigel having arbitrary form and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a method for producing a vitrigel having a form other than thin film form and to provide a vitrigel having various forms other than thin film form. <P>SOLUTION: A vitrigel having arbitrary form which cannot be produced by the conventional method comprising the removal of free water only by drying can be produced by processing a hydrogel to an arbitrary form before vitrification and introducing a free water removal step to use the gravity force in the vitrification step to perform the dehydration/drying of the hydrogel. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a structure having various shapes using vitrigel and a structure obtained thereby.

  After injecting the collagen sol into the culture dish and applying the optimal salt concentration, pH and temperature to gelation, it is thoroughly dried at low temperature and vitrified, and then rehydrated ( By rehydration), a technology has been established to convert the physical properties of collagen gel into a thin film with excellent strength and transparency with good reproducibility (Patent Document 1).

  Here, it has been reported that vitrification of heat-denatured protein proceeds in a process in which bound water gradually decreases after free water is removed in the drying step (Non-patent Document 1). If it is a hydrogel, gels of components other than collagen can be converted to stable new physical properties by rehydrating after vitrification, so new physical properties created through this vitrification process An academic term vitrigel was set as a term meaning a gel in a state (Non-patent Document 2).

However, the known methods for producing vitrigel can only produce hydrogels with a thin film shape as described in Patent Document 1, and cannot produce vitrigels with other shapes. It was. That is, when it is intended to produce a thin-film hydrogel by the method of removing free water by natural drying described in Patent Document 1, it takes a long time to remove free water. In this method, only a thin-film-shaped vitrigel that does not change its shape even when left standing for a long time can be produced. On the other hand, for example, when a columnar gel is placed in a culture dish to be dehydrated and vitrified, the cross-sectional shape changes greatly during the dehydration process, and the original gel shape cannot be recovered when rehydrated.
JP-A-8-228768 Takushi E, et al., Nature 345: 298-299, 1990 Takezawa T, et al., Cell Transplant. 13: 463-473, 2004

  This invention makes it a subject to develop the method of manufacturing vitrigel of shapes other than thin film shape. Another object of the present invention is to provide a vitrigel having various shapes other than a thin film shape.

  Free water by conventional drying only by introducing a free water removal process using gravity into the vitrification process where the hydrogel before vitrification is processed into an arbitrary shape and dehydrating and drying the hydrogel It was found that a vitrigel having an arbitrary shape that could not be produced in the removing step was produced.

  Specifically, the present invention dehydrates, dries and vitrifies a hydrogel having a desired shape other than a thin-film shape by setting the major axis direction of the hydrogel at an angle with respect to the horizontal. A method for producing a vitrigel having a desired shape is provided.

  In the present invention, a vitrigel having a desired shape other than a thin film shape can be produced by using the above method. The vitrigel produced in this way is biocompatible and low in immunogenicity, so it is widely used for medical purposes such as medical materials such as sutures and cell substrates (biomaterials) for biological repair in regenerative medicine. Can be used for

BEST MODE FOR CARRYING OUT THE INVENTION

  In the present invention, the hydrogel before vitrification is processed into an arbitrary shape, and a free water removal process using gravity is introduced into the vitrification process for drying the hydrogel, so that only conventional drying is performed. We succeeded in producing a vitrigel of arbitrary shape by greatly shortening the time of the free water removal process. The shape of vitrigel described in Patent Document 1 was only a thin film, but in the method using natural drying described, it takes a long time to remove free water. However, only a thin-film hydrogel that does not change its shape could be produced. Conversely, for example, when the hydrogel is vitrified according to the method described in Patent Document 1 in a state where the tubular hydrogel is laid on the plate, the cross-sectional shape is deformed. It was. And once it was vitrified in a deformed state, the desired shape could not be recovered even after rehydration.

  In exchange for this, the present invention dehydrates, dries and vitrifies a hydrogel having a desired shape other than a thin-film shape by setting the major axis direction of the hydrogel at an angle with respect to the horizontal. A method for producing a vitrigel having a desired shape is provided. In general, in the vitrification of hydrogel, first, the free water contained in the hydrogel is separated from the gel, and further, with the passage of time, the bonded water of the substance in the gel is removed and vitrified. Follow. On the other hand, as in the present invention, by setting the major axis direction of the hydrogel at an angle with respect to the horizontal, the free water can be removed much more than the method of Patent Document 1 described above. The required time can be shortened. As a result, the time required to vitrify the hydrogel can be remarkably shortened, and various shapes of vitrigels such as filaments, tubes, or rods can be produced. it can.

  In the present invention, when a desired shape other than the thin film shape is referred to, the cross section perpendicular to the major axis direction of the shape is deformed from the desired shape when the major axis direction of the shape is horizontal and dehydrated and dried to be vitrified. For example, a thread shape, a tubular shape, or a rod shape can be mentioned.

  In the method of the present invention, the vitrification step is performed by dehydrating and drying the long axis direction of the hydrogel at an angle with respect to the horizontal. By dehydrating and drying the long axis direction of the shape of the hydrogel at an angle with respect to the horizontal, glass is utilized more quickly and without deforming the cross-sectional shape using the difference in gravity generated in the long axis direction. Can be made. In this case, the angle of the hydrogel shape with respect to the horizontal in the major axis direction is preferably 60 to 90 degrees, more preferably 75 to 90 degrees, and most preferably 90 degrees.

  The composition of the hydrogel used as a raw material when producing vitrigel by vitrification of the present invention is not particularly limited as long as it is vitrified by dehydration and drying. The composition of hydrogel that can form vitrigel includes collagen such as type I collagen and type III collagen, or matrigel (base membrane component extracted from EHS tumor, mainly type IV collagen, laminin, and heparan sulfate proteoglycan) In addition, extracellular matrix components such as agar, agarose, acrylamide and the like are known. In the present invention, it is preferable to use collagen such as type I collagen, type III collagen, or a combination thereof.

  By adopting such a method, a vitrigel having a desired shape other than a thin film shape, which could not be produced because a cross-sectional structure perpendicular to the major axis direction has been distorted so far, is produced. Can do. The vitrigel prepared in this way is a rehydrated vitrified hydrogel in an aqueous medium. The vitrigel is not only rehydrated but also in a vitrified dry state before rehydration or Even in a dried state after rehydration and then drying again, it can be used depending on the application.

  For example, vitrigel made using collagen has the property of not causing an immune reaction in vivo, and at the same time has the feature of being absorbed in vivo. Therefore, the vitrigel produced in this way can be used for medical purposes. According to the method of the present invention, as long as a hydrogel having a desired shape can be produced, a vitrigel having various shapes can be produced. Therefore, a vitrigel according to the application can be provided.

  For example, in the case of a filamentous vitrigel, a surgical suture can be provided as a material in a dry state in a vitrified state before rehydration or in a dry state after being rehydrated and dried again. Such a suture is unlikely to cause an immune reaction in the living body as described above, and has a biocompatibility that it is absorbed in the living body. Therefore, the suture is an alternative to a conventionally used suture such as a cut gut. Can be used as

  In the present invention, it is also possible to provide an artificial blood vessel support made of tubular vitrigel. Since the vitrigel of the present invention can also be used as a cell culture base, first, endothelial cells are cultured on the inner surface of such an artificial blood vessel support, while smooth muscle cells are cultured on the outer surface. To produce a pseudo blood vessel and use it as an artificial blood vessel. This artificial blood vessel support functions as a scaffold for blood vessels in the body, has the property of promoting the regeneration of the blood vessel structure, and is biocompatible so that it can completely replace the blood vessels in the long term.

Example 1: Production of filamentous vitrigel In this example, a filamentous vitrigel was produced.

  First, at 4 ° C, an equal volume of 0.5% type I collagen aqueous solution (CELLGEN I-AC, manufactured by Koken Co., Ltd.) and culture solution (10% non-immobilized fetal bovine serum (FBS), 20 mM HEPES, 100 u / A 0.25% type I collagen sol was prepared by mixing with ml Penicillin and Dulbecco's Modified Eagle Medium (DMEM) containing 100 ig / ml streptomycin. After sealing the lower end of a 3 mm ID tube (1 ml standard pipette [Falcon # 7521]) with parafilm (Fig. 1A (a)), inject about 1.4 ml of the above 0.25% type I collagen sol. (FIG. 1A (b)). This was stored for 2 days or longer and gelled in a pipette (FIG. 1A (c)).

  After the gelation is completed, fix the top end of the 3 mm diameter collagen gel taken out from the pipette and hang it vertically. Free water is removed from the gel by its own weight in a thermostatic chamber at a temperature of 10 ° C and a humidity of 40%. It was drained (FIG. 1A (d)). Under the same conditions, the moisture (free water) in the gel is further removed using gravity (dehydration) (Fig. 1A (e)), and the moisture in the gel (free water and combined water) is further dried over time. (Vitrification) was performed (FIG. 1A (f)). Here, a decrease in gel volume reaches a plateau with the removal of water that was able to move freely in the gel (end of gel contraction), and a water-specific shine that can be judged when the gel is observed. Based on the loss, free water was completely removed and used as a measure of drying. The dried gel after completely removing free water is allowed to proceed to a vitrification process in which bound water is slowly removed while being stored at room temperature. In this example, in order to produce a filamentous vitrigel, 4 days as the gelation period, 1 day as the initial drying period to completely remove free water, and as the vitrification period to gradually remove the bound water It took 1 or 8 days. Thereafter, the filamentous vitrigel was completed by rehydration with PBS. In other words, the number of days from the time when the 0.25% type I collagen sol was made to the vitrified dry state before rehydrating the filamentous vitrigel (the period after fibrosis of collagen) is 6 days for the former The latter was 13 days.

  The state of initial drying of the filamentous vitrigel prepared in this manner is shown in FIG. 1B.

Example 2 Production of Tubular Vitrigel In this example, a tubular vitrigel was produced.

  Insert a 0.25% type I collagen sol prepared in the same manner as in Example 1 into a cylinder (II) with an outer diameter of 4 mm so that it is concentric inside the tube (I) with an inner diameter of 8 mm, and the lower end is parafilm. After sealing (FIG. 2A (a)), about 8 ml of 0.25% type I collagen sol was injected into the annular columnar gap composed of I and II (FIG. 2A (b)). This was kept at 37 ° C. and gelled into a tubular shape in the tube (FIG. 2A (c)).

  After the gelation is completed, remove the parafilm and cylinder (II) at the lower end and keep the gel in the outer tube (I) in a constant temperature and humidity chamber at a temperature of 10 ° C and a humidity of 40%. The tube (I) was fixed vertically, and free water flowed out of the gel by its own weight (FIG. 2A (d)). Under the same conditions, the moisture (free water) in the gel is further removed using gravity (dehydration) (Figure 2A (e)), and the moisture (free water and combined water) in the gel is dried over time. (Vitrification) was performed for about 2 weeks (FIG. 2A (f)).

  The tubular vitrigel thus prepared was rehydrated with PBS. The tubular vitrigel thus produced is shown in FIG. 2B.

Example 3: Production of rod-shaped vitrigel In this example, a cap-like (short rod-shaped) vitrigel was produced.

  About 1 ml of a 0.25% type I collagen sol prepared in the same manner as in Example 1 was injected inside a glass round bottom test tube having an inner diameter of 10 mm (FIG. 3 (b)). This was kept at 37 ° C. and gelled into a short rod in the tube (FIG. 3 (c)).

  After gelation is completed, remove the free water from the supernatant by centrifuging (1,400 g x 10 min), and then invert the test tube in a constant temperature and humidity chamber at a temperature of 10 ° C and a humidity of 40%. The free water was allowed to flow out of the gel by its own weight (Fig. 3 (d)). Under the same conditions, the moisture (free water) in the gel was further removed using gravity (dehydration), and the moisture (free water and combined water) in the gel was dried (vitrified) over time. (Figure 3 (e)). The tubular vitrigel thus prepared was rehydrated with PBS and used.

Example 4: Examination of applicability of filamentous vitrigel to medical use In this example, the applicability of the filamentous vitrigel prepared in Example 1 to medical use was examined.

  In the case of the filamentous vitrigel prepared in Example 1 by the method of the present invention, in particular, the vitrified dry state before rehydration or the dry state material dried again after rehydration is used as a suture. Since the use was considered, first of all, 3-0 silk suture (Blade Silk, Natsume Seisakusho Co., Ltd., nominal diameter 0.27 mm), 4-0 silk suture (Black Blade Silk, Inc.) ) Natsume Seisakusho, nominal diameter 0.20 mm), 5-0 silk suture (soft white blade silk thread, Akiyama Seisakusho, nominal diameter 0.15 mm), 3-0 biodegradable suture (Coated VICRYL, ETHICON, Inc) ., Nominal diameter 0.27 mm).

  Vitrified dry material before rehydrating the filamentous vitrigel prepared in Example 1 (hereinafter referred to as “filamentous vitrigel (dry state)” in this example) (FIGS. 4A and B), Dry forms of 5-0 silk suture (Figure 4C), 4-0 silk suture (Figure 4D), 3-0 silk suture (Figure 4E), and 3-0 biodegradable suture (Figure 4F) Were compared under a microscope. As can be seen from these photographs, a filamentous vitrigel (in the dry state) (Figs. 4A and B) of approximately the same thickness (0.15 mm in diameter) as the 5-0 silk suture (Fig. 4C) has been produced. It was revealed. Also, it was revealed that the surface was smoothly formed, unlike silk thread.

  Next, the strength of the filamentous vitrigel (dried state) produced in Example 1 was measured in comparison with a 5-0 silk suture of approximately the same thickness, using the breaking strength and the stretch length at break as indices. Specifically, the strength of the filamentous vitrigel (dry state) was measured using a digital force gauge (manufactured by Nidec Simpo Co., Ltd.), after cutting to a total length of 5 cm, fixing 1 cm at both ends, The breaking strength (Kgf) when pulled at 9 mm per minute and the extension length (mm) at breaking were measured. In FIGS. 5A and 5B, lane 1 is a 6-day filamentous vitrigel (dried state); lane 2 is a 13-day filamentous vitrigel (dried state); and lane 3 is 5-0 silk suture data, respectively. Show. As shown in FIGS. 5A and 5B, the filamentous vitrigel (dried state) with 13 days of preparation (lane 2) has almost the same breaking strength as the 5-0 silk suture (lane 3) with the same thickness. (About 400 gf), and the extension length at break was about 3.4 mm, which was greater than about 2.6 mm of 5-0 silk suture.

  Furthermore, for the purpose of showing that such strength is sufficiently tolerable for surgery, a suture experiment of rat epidermis was performed using a filamentous vitrigel (dried state) with a production period of 6 days ( Figure 6). The left side of FIG. 6 is a photograph on the day when the suture is performed, and the right side of FIG. 6 is a photograph one day after the suture is performed. As can be seen from this experiment, it was found that when the rat epidermis was sutured, it exhibited strength as a suture.

  In the present invention, a vitrigel having a desired shape other than a thin film shape can be produced by using the above method. The vitrigel produced in this way is biocompatible and low in immunogenicity, so it is widely used for medical purposes such as medical materials such as sutures and cell substrates (biomaterials) for biological repair in regenerative medicine. Can be used for

FIG. 1 shows a procedure for producing a filamentous vitrigel and a photograph of the filamentous vitrigel produced. FIG. 2 shows a procedure for producing a tubular vitrigel and a photograph of the produced tubular vitrigel. FIG. 3 shows a procedure for producing a cap-like (short rod-like) vitrigel. Figure 4 shows 5-0 silk suture (Figure 4C), 4-0 silk suture (Figure 4D), 3-0 silk suture (Figure 4E), and 3-0 biodegradable suture (Figure 4F) FIG. 4 shows micrographs of the filamentous vitrigel of Example 1 (dried state) (FIGS. 4A and B) compared with the dried form of FIG. FIG. 5 shows the breaking strength (Kgf) (FIG. 5A) and extension length (mm) at break (FIG. 5B) of the filamentous vitrigel of Example 1 (dry state) compared to 5-0 silk suture. FIG. 6 shows photographs at the time of suturing (left) and 1 day after suturing (right) when the rat epidermis was sutured using the filamentous vitrigel of Example 1 (dried state).

Claims (15)

  Desired shape, characterized in that a hydrogel having a desired shape other than a thin film shape is vitrified by dehydration and drying by setting the major axis direction of the hydrogel at an angle to the horizontal. Of manufacturing vitrigel.   2. The method for producing a vitrigel according to claim 1, wherein the desired shape other than the thin film shape is a thread shape, a tubular shape, or a rod shape.   The method for producing vitrigel according to claim 1 or 2, wherein the vitrification is performed by setting the major axis direction of the hydrogel at an angle of 60 to 90 degrees with respect to the horizontal.   The method for producing a vitrigel according to any one of claims 1 to 3, wherein the hydrogel is collagen.   5. The method for producing a vitrigel according to claim 4, wherein the collagen is formed from type I collagen, type III collagen, or a combination thereof.   Vitrigel having a desired shape other than a thin film shape.   7. The vitrigel according to claim 6, wherein the desired shape other than the thin film shape is a thread shape, a tubular shape, or a rod shape.   The vitrigel according to claim 6 or 7, wherein a cross section perpendicular to the major axis direction of the shape is a circle or a concentric circle.   The vitrigel according to any one of claims 6 to 8, wherein the collagen gel is vitrified.   10. The vitrigel according to claim 9, wherein the collagen is formed from type I collagen, type III collagen, or a combination thereof.   The vitrigel according to any one of claims 6 to 10, for use in medicine.   Sutures made from thread-like vitrigel.   The suture of claim 12, wherein the suture is biocompatible.   An artificial blood vessel support made of tubular vitrigel. 15. The artificial blood vessel support according to claim 14, which is biocompatible.